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Am J Physiol Heart Circ Physiol 294: H1906-H1913, 2008. First published February 1, 2008; doi:10.1152/ajpheart.00966.2006
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Key role of {alpha}1β1-integrin in the activation of PI3-kinase-Akt by flow (shear stress) in resistance arteries

Laurent Loufrani,1 Kevin Retailleau,1 Arnaud Bocquet,1 Odile Dumont,1 Kerstin Danker,2 Huguette Louis,3 Patrick Lacolley,3 and Daniel Henrion2

1Centre National de la Recherche Scientifique UMR 6214, Institut National de la Santé et de la Recherche Médicale (INSERM) 771, University of Angers, Angers and 3INSERM U684, Nancy, France; and 2University Medical Center Charité, Berlin, Germany

Submitted 6 September 2006 ; accepted in final form 24 January 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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Resistance arteries are the site of the earliest manifestations of many cardiovascular and metabolic diseases. Flow (shear stress) is the main physiological stimulus for the endothelium through the activation of vasodilatory pathways generating flow-mediated dilation (FMD). The role of FMD in local blood flow control and angiogenesis is well established, and alterations in FMD are early markers of cardiovascular disorders. {alpha}1-Integrin, which has a role in angiogenesis, could be involved in FMD. FMD was studied in mesenteric resistance arteries (MRA) isolated in arteriographs. The role of {alpha}1-integrins in FMD was tested with selective antibodies and mice lacking the gene encoding for {alpha}1-integrins. Both anti-{alpha}1 blocking antibodies and genetic deficiency in {alpha}1-integrin in mice ({alpha}1–/–) inhibited FMD without affecting receptor-mediated (acetylcholine) endothelium-dependent dilation or endothelium-independent dilation (sodium nitroprusside). Similarly, vasoconstrictor tone (myogenic tone and phenylephrine-induced contraction) was not affected. In MRA phosphorylated Akt and phosphatidylinositol 3-kinase (PI3-kinase) were significantly lower in {alpha}1–/– mice than in {alpha}1+/+ mice, although total Akt and endothelial nitric oxide synthase (eNOS) were not affected. Pharmacological blockade of PI3-kinase-Akt pathway with LY-294002 inhibited FMD. This inhibitory effect of LY-294002 was significantly lower in {alpha}1–/– mice than in {alpha}1+/+ mice. Thus {alpha}1-integrin has a key role in flow (shear stress)-dependent vasodilation in resistance arteries by transmitting the signal to eNOS through activation of PI3-kinase and Akt. Because of the central role of flow (shear stress) activation of the endothelium in vascular disorders, this finding opens new perspectives in the pathophysiology of the microcirculation and provides new therapeutic targets.

myogenic tone; nitric oxide; NG-nitro-L-arginine methyl ester; Akt; {alpha}1-integrin; transgenic mice; mechanotransduction

RESISTANCE ARTERIES CONTROL blood flow to tissues. They possess a basal tone, mainly of myogenic and sympathetic origin, allowing rapid changes in diameter and consequently rapid changes in blood flow. Resistance artery tone is regulated by mechanical (tensile stress due to pressure and shear stress due to flow), chemical, and neurohormonal factors. Pressure and flow are the main determinants of basal tone in resistance arteries and thus allow a rapid adaptation to changes in flow and pressure in order to maintain optimal tissue perfusion (4). Pressure-induced contraction (myogenic tone) is a characteristic of small resistance arteries, determining in synergy with the other vasoactive systems a sustained tone in resistance arteries (4, 10, 13). This basal tone is opposed by flow (shear stress)-mediated dilation (FMD), in vitro as well as in vivo (4, 12, 13). Flow stimulates endothelial cells continuously, and changes in sensitivity to flow (shear stress) have dramatic consequences in cardiovascular and metabolic diseases.

Flow (shear stress) activates extracellular elements and adhesion sites (12) linking the cytoplasmic tails of integrins with cytoskeletal proteins. Integrins such as {alpha}vβ5-, {alpha}vβ3-, or {alpha}5β1-integrins, which are receptors for fibronectin, are involved in cell attachment and migration (12, 44, 46, 47, 49). They are important for vasculogenesis and angiogenesis (2, 18, 19, 43). Nevertheless, although {alpha}1-integrins are involved in angiogenesis (23, 39), endothelial cell migration (40), and tumor vascularization(37), their role in flow sensing, important for these phenomena, is not yet clearly described.

{alpha}1β1-Integrin belongs to the family of receptors for collagen and laminin. {alpha}1β1-Integrin is abundant at the surface of microvascular endothelial cells (15), where it might be involved in flow mechanotransduction. In cultured cells from human umbilical vein, flow induces the phosphorylation of Akt through activation of fibronectin-binding integrins (16). Furthermore, flow activates phosphatidylinositol 3-kinase (PI3-kinase), Akt, and endothelial nitric oxide synthase (eNOS) via src kinase (28).

Thus we hypothesize that {alpha}1β1-integrin might play a role in FMD via activation of PI3-kinase and Akt to stimulate eNOS in small resistance-sized arteries, such as the mesenteric artery, and large-conduit arteries, such as the carotid artery, in wild-type mice and mice deficient for {alpha}1-integrin.


   METHODS
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RESULTS
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Animals

In a first series of experiments, adult male Wistar rats (Iffa-Credo, L'Arbresle, France) were anesthetized (pentobarbital sodium, 50 mg/kg ip), and mesentery was removed in order to isolate mesenteric resistance arteries (MRA). In another series of experiments, 20-wk-old {alpha}1-integrin-deficient ({alpha}1–/–) mice and their littermate controls ({alpha}1+/+) were used. A null mutation in the {alpha}1-integrin in mice was described previously by Gardner et al. (21). All procedures were performed in accordance with institutional guidelines for animal experimentation under Authorization No. 49045(6422), Veterinary Department (Préfecture of Maine-et-Loire).

Arterial Pressure Measurement

After anesthesia (pentobarbital sodium, 5 mg/kg ip) the left carotid artery was cannulated in order to measure blood pressure (30). The cannula was connected to a pressure transducer (Gould P10EZ, Spectramed, Oxnard, CA), and the signal was recorded (Biopac, La Jolla, CA). The mesentery was subsequently removed for dissection and MRA sampling.

Pressure and Flow-Dependent Tone in Carotid Arteries and Mesenteric Resistance Arteries

A segment of third-order mesenteric artery (~150 µm) or carotid artery was cannulated at both ends and mounted in a video-monitored perfusion system (24) as previously described (25). Briefly, arterial segments were bathed in a 5-ml organ chamber containing a physiological salt solution (PSS) of the following composition (in mmol/l): 135.0 NaCl, 15.0 NaHCO3, 4.6 KCl, 1.5 CaCl2, 1.2 MgSO4, 11.0, glucose, and 10.0 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. pH was maintained at 7.4, PO2 at 160 mmHg, and PCO2 at 37 mmHg. The chamber was superfused at a rate of 2.5 ml/min. Arterial diameter was measured and recorded continuously with a video monitoring system (Living System Instrumentation, Burlington, VT). Pressure and flow rate could be changed independently. To determine myogenic tone, equilibrium diameter changes were measured in each segment when intraluminal pressure was set at 25, 50, 75, 100, and 125 mmHg. To determine FMD at 75 mmHg of pressure, arteries were contracted with phenylephrine (PE) at 50% of non-receptor-dependent contraction (KCl, 80 mmol/l) and submitted to intraluminal flow increased stepwise (0–100 µl/min for mesenteric arteries and 0–800 µl/min for carotid arteries). This was subsequently repeated after addition of NG-nitro-L-arginine methyl ester (L-NAME, 100 µmol/l) or LY-294002 (10 µmol/l) to the PSS. At the end of each experiment arteries were perfused and superfused with a Ca2+-free PSS containing ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, 2 mmol/l) and sodium nitroprusside (SNP, 100 µmol/l). Pressure steps (25–150 mmHg) were then repeated in order to determine the passive diameter (PD) of the vessel in the absence of smooth muscle tone. Diameter measurements made in normal PSS were considered as diameter under active tone (or active diameter). Pressure and diameter measurements were continuously collected for analysis (Biopac MP 100).

Myogenic tone was calculated as percentage of PD (25). FMD was expressed as percent dilation of myogenic tone (25). Under steady-state conditions wall shear stress (dyn/cm2) was calculated as previously described (25).

The experiments described above were also performed in rat mesenteric arteries in the presence or absence of anti-{alpha}1 antibodies (and with the nonactive anti-{alpha}1 antibody) and in mesenteric arteries isolated from {alpha}1–/– or {alpha}1+/+ mice. The neutralizing antibody was graciously given by Dr. K. Dankes (University Medical Center Charité). The antibody was added to the intra- and extraluminal solutions at a concentration of 5 µg/ml (30 min).

Pharmacological Properties of Resistance Arteries

Segments of mesenteric arteries (2 mm long) were dissected and mounted on a wire myograph (DMT, Aarhus, Denmark). Briefly, two tungsten wires (25-µm diameter) were inserted into the lumen of the arteries and fixed to a force transducer and a micrometer, respectively. Arteries were bathed in PSS, described above, maintained at 37°C and pH 7.4 (PO2 160 mmHg, PCO2 37 mmHg). Wall tension was applied as described previously (35). Vessels were then allowed to stabilize for 1 h. Artery viability was tested with a potassium-rich solution (80 mmol/l PSS). Endothelium was considered functional when an 80% ACh-induced relaxation (10–6 mol/l) was obtained after PE-induced preconstriction (10–6 mol/l). A cumulative concentration-response curve (CRC) to PE (10–9–10–4 mol/l) was performed. After washout, CRCs to ACh (10–9–10–5 mol/l) were constructed after PE-induced preconstriction (10–6 mol/l). This protocol ended with a CRC to SNP (10–9–10–5 mol/l) after PE-induced preconstriction (10–6 mol/l).

In a separate series of experiments, CRCs were obtained to PE (0.01 nmol/l–1 µmol/l) (30). ACh and SNP CRCs were obtained after preconstriction of the artery with PE (1 µmol/l) (31).

Immunofluorescence Analysis of {alpha}1-Integrin

Segments of MRA were mounted in embedding medium (Miles), frozen in isopentane precooled in liquid nitrogen, and stored at –80°C. Immunostaining of {alpha}1-integrin was performed on transverse 7-µm-thick cross sections incubated with anti-{alpha}1 antibodies (1:100 in PBS overnight incubation, 4°C) and with anti-rabbit antibodies labeled with FITC (1:200 in PBS, 90 min at 37°C). Positive staining was visualized with confocal microscopy and QED-image software (Solamere Technology, Salt Lake City, UT).

Western Blot Analysis of MRA

Segments of MRA were isolated from {alpha}1–/– or {alpha}1+/+ mice just after arterial pressure measurement as described above. The arteries were quickly frozen in a physiological condition of pressure of flow (in vivo shear stress) and stored at –80°C before analysis. Arterial segments were then homogenized (Ultrasonic Processor, Bioblock Scientific). Proteins were separated by SDS-PAGE (Mini gel protean II system, Bio-Rad; 100 V, using 300 ml of 25 mmol/l Tris, 192 mmol/l glycine, 0.1% SDS) using a 4% stacking gel followed by a 7% running gel. After migration, proteins were transferred (100 V, 1 h 30 min, 4°C, using 800 ml of 25 mmol/l Tris, 192 mmol/l glycine, 10% methanol) to polyvinylidene difluoride blotting membranes (Immobilon-P, Millipore). Membranes were then washed in TBS-T buffer (composition: 10 mmol/l Tris-base pH 7.5, 0.1 mol/l NaCl, 1 mmol/l EDTA, 0.1% Tween 20) and blocked for 2 h at room temperature (5% fat-free dry milk in TBS-T). Membranes were incubated overnight at 4°C with the primary antibody against phospho-Akt, Akt, eNOS, PI3-kinase or {alpha}1-integrin (BioLabs and Santa Cruz Biotechnology), washed again, and incubated with horseradish peroxidase-conjugated secondary antibody (Amersham, 90 min at room temperature, 1/2,000). Membranes were washed, and Akt, phospho-Akt, eNOS, {alpha}1-integrin, and actin bands were visualized and quantified (ECL-Plus, Amersham).

Statistical Analysis

Results are expressed as means ± SE. Significance of the differences between groups was determined by analysis of variance (1-factor ANOVA or ANOVA for consecutive measurements, when appropriate) or unpaired Student's t-test. P values <0.05 are considered to be significant.


   RESULTS
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{alpha}1-Integrin was visualized in MRA with immunostaining and confocal microscopy. {alpha}1-Integrins were mainly detected in the endothelium of MRA isolated from control mice (Fig. 1A) or rats (data not shown) but not in integrin-deficient mice (Fig. 1B).


Figure 1
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  		Fig. 1. Immunolocalization of {alpha}1-integrin (arrows) by confocal microscopy in mesenteric resistance arteries (MRA) isolated from {alpha}1+/+ (A) and {alpha}1–/– (B) mice.

 
Role of {alpha}1-Integrin in Rats

In isolated rat MRA submitted to stepwise increases in intraluminal pressure, myogenic tone developed. Myogenic tone was not affected by anti-{alpha}1-integrin antibodies (for example, when pressure was 100 mmHg, myogenic tone was 35 ± 5% of PD in control conditions and 37 ± 6% of PD in the presence of anti-{alpha}1-integrin antibodies). Stepwise increases in flow (shear stress) induced a dilation that was significantly attenuated by anti-{alpha}1-integrin antibodies (Fig. 2) but not by the nonactive anti-{alpha}1-integrin antibodies (data not shown).


Figure 2
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  		Fig. 2. Changes in diameter in response to stepwise increases in shear stress in MRA pretreated with anti-{alpha}1 antibodies or not (control) (n = 6/group). *P < 0.01, anti-{alpha}1 vs. control.

 
Phenylephrine, ACh, and SNP, inducing concentration-dependent contraction or dilation, in isolated MRA were not affected by anti-{alpha}1-integrin antibodies (Table 1).


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  		Table 1. Contraction to PE and dilation to ACh and SNP in rat mesenteric resistance arteries treated with anti-{alpha}1-integrin antibodies or not (control)

 
Role of {alpha}1-Integrin in Mice

The absence of {alpha}1-integrin in mice did not affect mouse body weight (32 ± 1 vs. 33 ± 1 g, {alpha}1–/– vs. control mice; n = 7/group). Mean arterial blood pressure, measured in the left carotid artery under anesthesia, was slightly but not significantly higher in {alpha}1–/– mice (mean arterial pressure 109 ± 8 mmHg; n = 7) than in {alpha}1+/+ mice (97 ± 5 mmHg; n = 7).

Carotid and Mesenteric Resistance Arteries

Mechanotransduction and pharmacological results. In MRA and carotid arteries isolated from {alpha}1–/– and {alpha}1+/+ mice myogenic tone was not affected by the absence of {alpha}1-integrin (Fig. 3), but FMD was significantly lower in MRA from {alpha}1–/– mice than in control ({alpha}1+/+) mice (Fig. 4). The maximal dilation induced by flow in MRA was decreased by 52% in {alpha}1–/– mice compared with {alpha}1+/+ mice (Fig. 4B). In MRA and carotid arteries, nitric oxide (NO) synthesis blockade with L-NAME reduced FMD (Fig. 4B). The reduction in FMD induced by L-NAME was not significantly different in {alpha}1–/– (60 ± 8% decrease in maximal dilation) and {alpha}1+/+ (56 ± 4%) mice in mesenteric arteries.


Figure 3
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  		Fig. 3. A: representative curve of myogenic tone in response to pressure in MRA isolated from {alpha}1–/– and {alpha}1+/+ mice. B: myogenic tone in response to pressure in MRA isolated from {alpha}1–/– and {alpha}1+/+ mice (n = 7/group). C: myogenic tone and passive diameter in response to pressure (100 mmHg) in carotid arteries and MRA isolated from {alpha}1–/– and {alpha}1+/+ mice (n = 7/group).

 

Figure 4
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  		Fig. 4. A: representative curve of flow- mediated dilation in response to shear in MRA isolated from {alpha}1–/– and {alpha}1+/+ mice. B: changes in diameter in response to increases in shear stress (75 dyn/cm2) in carotid arteries and MRA isolated from {alpha}1–/– and {alpha}1+/+ mice with and without NG-nitro-L-arginine methyl ester (L-NAME, 0.1 mM; n = 7/group). *P < 0.01, #P < 0.01, effect of L-NAME.

 
Passive arterial diameter was not affected by the absence of {alpha}1-integrin in carotid and mesenteric arteries (Fig. 3; PD in MRA was 217 ± 7, 240 ± 5, and 253 ± 6 µm in {alpha}1+/+ mice vs. 221 ± 11, 231 ± 11, and 238 ± 10 µm in {alpha}1–/– mice for pressure values of 75, 100, and 125 mmHg, respectively; n = 7/group).

PE-induced contraction as well as ACh- and SNP-dependent dilation were not modified in {alpha}1–/– compared with {alpha}1+/+ mice (Table 2).


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  		Table 2. Contraction to PE and dilation to ACh and SNP in mesenteric resistance arteries from mice lacking gene for {alpha}1-integrin and control mice

 
Biochemical results. The expression of eNOS and Akt did not change in MRA from {alpha}1–/– compared with wild-type mice, but phospho-Akt and PI3-kinase were significantly lower in {alpha}1–/– compared with control mice (Fig. 5, A and B). In {alpha}1–/– mice, {alpha}1-integrin was not found in MRA by either Western blot analysis or immunostaining (data not shown).


Figure 5
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  		Fig. 5. A and B: expression of {alpha}1-integrin, endothelial nitric oxide synthase (eNOS), Akt, phospho-Akt, phosphatidylinositol 3-kinase (PI3-kinase), and actin in MRA isolated from {alpha}1–/– and {alpha}1+/+ mice (A) and average of 6 different experiments (B). C and D: changes in diameter (flow-mediated dilation) in response to stepwise increases in shear stress in MRA isolated from {alpha}1–/– (D) and {alpha}1+/+ (C) mice after pretreatment of the arteries for 30 min with the PI3-kinase inhibitor LY-294002 (n = 6/group). *P < 0.01, {alpha}1+/+ vs. {alpha}1–/– mice; #P < 0.01, effect of LY-294002.

 
The involvement of the PI3-kinase-Akt pathway in FMD was then tested functionally in MRA isolated from {alpha}1+/+ and {alpha}1–/– mice. The inhibitor LY-294002 (10 µmol/l) significantly reduced FMD in control mice. The inhibitory effect of LY-294002 (10 µmol/l) on FMD was significantly lower in {alpha}1–/– than in {alpha}1+/+ mice (Fig. 5, C and D).


   DISCUSSION
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 METHODS
 RESULTS
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GRANTS
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This study identifies for the first time {alpha}1-integrin as a key element in FMD in resistance arteries and carotids. Indeed, both antibodies directed against {alpha}1-integrins and the lack of the gene encoding for {alpha}1-integrins selectively and highly reduced FMD in MRA. Pharmacological responses to dilatory and contractile agents as well as myogenic tone due to pressure were not affected. In addition, {alpha}1-integrin was functionally linked to the PI3-kinase-Akt pathway, mediating the acute response to flow (shear stress) in resistance arteries.

Integrins are heterodimeric adhesion receptors (27) for collagens and laminins consisting of {alpha}- and β-subunits capable of binding extracellular matrix molecules as well as other adhesion receptors on neighboring cells (6). Integrins participate in cell migration, tissue organization, cell growth, homeostasis, inflammation, target recognition, and differentiation of many cell types (5, 7, 41, 42). {alpha}1-Integrin belongs to the β1-binding family of integrins (41, 42). They are widely expressed in the adult and are found in visceral and some vascular smooth muscle, liver, microvascular endothelium, activated lymphocytes, and dermis (9, 21). Although this integrin is important for endothelial cell migration and angiogenesis, its possible role in flow (shear stress)-induced dilation has not been investigated in arteries and especially in resistance arteries, the site of the earliest manifestations of cardiovascular and metabolic diseases.

The mechanism by which hemodynamic forces, such as shear stress and pressure, are transduced into cellular signaling is still not fully elucidated, especially in small arteries submitted to physiological hemodynamic forces. In vivo, resistance arteries submitted to mechanical forces (shear stress and pressure) develop a highly autoregulated tone in order to adapt local blood flow supply to organ needs (4, 13, 29). FMD and pressure-induced myogenic tone have a key role in the control of resistance arteries tone and consequently in the control of local blood flow (1).

We found that {alpha}1-integrin was expressed in resistance arteries of rats and wild-type mice. It was mainly expressed at the level of endothelial cells, in agreement with previous observations (15, 23). Although a positive staining of {alpha}1-integrins was also seen in the smooth muscle cell layer, neither the blocking antibody nor the knockout affected artery contractility or dilation with a NO donor. Thus {alpha}1-integrin, although present in the media, is not directly involved in acute responses to vasoactive agents. Contractions due to adrenergic receptor activation or myogenic tone were not affected in {alpha}1–/– mice. In previous studies, we showed that myogenic tone is unaffected by the absence of the cytoskeletal proteins vimentin (26, 45), desmin (31), or dystrophin (30). Indeed, myogenic tone and agonist (norepinephrine, angiotensin II)-induced tone might preferentially involve other integrins, such as {alpha}vβ5- or {alpha}4β1-integrins (14, 33, 38, 48, 51), and ultimately a cytoskeletal rearrangement, mainly through an increase in the ratio of F- to G-actin (8).

The main finding of the present study is that flow (shear stress)-induced dilation was selectively and strongly attenuated in resistance arteries by anti-{alpha}1-integrin antibodies and in {alpha}1–/– mice in mesenteric and carotid arteries. This effect was selective, because both endothelium-dependent (ACh) and endothelium-independent (SNP) dilation were not affected by the absence of {alpha}1-integrin or by anti-{alpha}1 antibodies. Thus the dilatory machinery, depending on the endothelium (ACh) or on the smooth muscle (SNP), was not affected by the blockade or absence of {alpha}1-integrin. Furthermore, PD was not affected by the absence of {alpha}1-integrin. This suggests that impairment in acute FMD does not lead to impairment in structural integrity (arterial lumen diameter as in arterial remodeling). In addition, eNOS expression was not altered in {alpha}1-integrin-deficient mice, further supporting the absence of change in the endothelial capacity to produce vasodilator agents. Only flow (shear stress)-dependent dilation was decreased. Thus we can postulate that {alpha}1-integrin is selectively involved in shear stress-dependent mechanotransduction leading to vasodilation in MRA. The role of integrins in FMD has previously been suggested in rat coronary arteries, in which {alpha}3β5-integrin blockade with selective RGD peptides or antibodies decreases FMD (34). Similarly, flow-induced contraction in rat cerebral arteries involves {alpha}3β5-integrin (32). Thus, depending on the vessel type and possibly on the species, different integrins may be involved in FMD. The increase in blood pressure, statistically nonsignificant, found in {alpha}1–/– mice supports the assumption that FMD has a key role in vascular homeostasis and in the control of blood pressure. In most cardiovascular diseases a decrease in FMD is the hallmark of an endothelium dysfunction.

Because endothelium-derived NO is the primary relaxing factor in MRA (26, 30), we tested the effect of NO synthesis blockade with L-NAME on FMD. L-NAME decreased FMD similarly in both {alpha}1–/– and {alpha}1+/+ mice, and eNOS expression was not affected by the absence of {alpha}1-integrins. These experiments suggest that the absence of {alpha}1-integrin impaired the flow-sensing process upstream of endothelial NO synthesis.

Phosphorylated Akt and PI3-kinase expression were decreased in resistance arteries from {alpha}1–/– mice compared with control mice. In addition, FMD was strongly inhibited after pharmacological inhibition of the PI3-kinase-Akt pathway in control mice but not in {alpha}1–/– mice. Thus this study provides the first evidence that Akt is involved in flow (shear stress)-induced vasodilation. Akt serves as a multifunctional regulator of cell survival and growth glucose metabolism (11) and as an activator of endothelial cell NO production in response to shear stress, through its ability to phosphorylate eNOS (16, 20). Nevertheless, these previous studies involved cells cultured in static conditions and then submitted to flow for at least several hours. This approach is of importance in the understanding of long-term changes induced by flow in large vessels prone to develop atherosclerosis. In the control of arterial diameter, and thus in the control of local blood flow, changes in flow occur rapidly, inducing immediate responses of the endothelium leading to diameter changes. Our results suggest that such changes in blood flow (shear stress) in the microcirculation stimulate PI3-kinase, Akt, eNOS, and NO production. This activation of the endothelium by flow is, at least in part, mediated by {alpha}1β1-integrin.

Interestingly, our data might also help in discerning two functional pathways leading to NO production by activation of eNOS. Indeed, on one hand eNOS expression and the endothelial capacity to dilate MRA were not affected (Ach-induced dilation in {alpha}1–/– mice or in the presence of anti-{alpha}1 antibodies). On the other hand, FMD and Akt phosphorylation were strongly decreased in {alpha}1–/– mice, suggesting that FMD involved a pathway different from receptor (Ach)-mediated dilation. This is in agreement with the previous data obtained in isolated cells (16, 20).

{alpha}1-Integrin has been involved in several diseases such as genetic disorder (Alport syndrome), graft-vs.-host diseases, and chronic stages of myocardial infarction (3, 9, 36). In addition, disorders in resistance artery function have been shown in most cardiovascular diseases such as hypertension, in ischemic diseases, and in diabetes and cancer, and a decrease in FMD is the hallmark of all these cardiovascular and metabolic diseases (1, 17, 50). Thus the present study, by bringing new insight into the mechanism of flow (shear stress) mechanotransduction in resistance arteries, opens new perspectives in the understanding of these diseases.

In conclusion, our study demonstrates that {alpha}1-integrin has a key role in flow (shear stress)-induced vasodilation in MRA. The selective activation by flow (shear stress) of the PI3-kinase-Akt-NOS pathway is mediated, at least in part, by {alpha}1-integrin.


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This work was supported in part by a grant from the French Association against Myopathies (AFM: Association France-Myopathies), Paris, France.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. Loufrani, UMR CNRS-INSERM 6214-771, Faculté de Medecine, 49045 Angers, France (e-mail: laurent.loufrani{at}wanadoo.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.


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