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Cardiovascular Research 2006 72(2):349-357; doi:10.1016/j.cardiores.2006.08.008
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Copyright © 2006, European Society of Cardiology

Carotid arterial stiffness, elastic fibre network and vasoreactivity in semicarbazide-sensitive amine-oxidase null mouse

Nathalie Merciera,b, Mary Osborne-Pellegrinc, Khadija El Hadrid, Augustine Kakoua, Carlos Labata, Laurent Loufranie, Daniel Henrione, Pascal Challandef, Sirpa Jalkanenb, Bruno Fèveg and Patrick Lacolleya,*

aINSERM U684, Vandoeuvre-les-Nancy, France
bMediCity Research Laboratory, University of Turku and National Public Health Institute, Turku, Finland
cINSERM U698, Paris, France
dUMR 7079 CNRS, Paris, France
eUMR-CNRS 6188, Angers, France
fUniversité Pierre et Marie Curie-Paris 6, FRE 2867 Saint-Cyr-l'Ecole, France
gINSERM U693, Le Kremlin-Bicêtre, France

* Corresponding author. INSERM U684, Faculté de Médecine, 9 Avenue de la Forêt de Haye, BP 184, 54505 Vandoeuvre-les-Nancy, Cedex, France. Tel.: +33 3 83 68 36 23; fax: +33 3 83 68 36 39. Email address: patrick.lacolley{at}nancy.inserm.fr

Received 1 April 2006; revised 28 July 2006; accepted 13 August 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: We examined the arterial phenotype of semicarbazide-sensitive amine-oxidase null mouse (SSAO –/–) using various techniques including high resolution echotracking.

Methods and results: SSAO –/– mice showed no change in arterial pressure under anesthesia. The in vivo arterial diameter, only measured in the carotid artery (CA), was higher in SSAO –/– than in SSAO +/+ animals. Elastic modulus–wall stress curves and CA rupture pressure were similar between SSAO –/– and +/+ mice, indicating no change in arterial wall stiffness or mechanical strength. There was no significant difference in insoluble elastin, total collagen content and elastic lamellar morphology between the two genotypes. No alteration in vascular reactivity was observed in aortic rings and mesenteric arteries from SSAO –/– mice. Aortic lysyl oxidase (LO) activity remained unaltered, indicating that SSAO invalidation is not accompanied by a compensatory increase in LO activity.

Conclusion: This is the first functional study of arteries lacking SSAO. Our results indicate that SSAO –/– mice present an increased arterial diameter associated with normal arterial mechanical properties, suggesting that SSAO deficiency might contribute to arterial wall remodeling. However, these results argue against the hypothesis that SSAO intervenes in elastic fibre organization, elastin cross-linking processes and vasoreactivity.

KEYWORDS Amine oxidase; Arterial stiffness; Elastin; Gene invalidation; SSAO; Vascular smooth muscle cells


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Aneurysms may result from the destruction of the extracellular matrix (ECM) proteins via inflammatory proteases, or from an alteration in elastic fibre organization, including quantitative and/or qualitative changes in some molecular components of elastic or collagen fibres [1]. Amine oxidases synthesized by vascular smooth muscle cells (VSMCs) are considered to exert a key role in ECM cross-linking processes [2,3]. Apart from atherosclerotic or inflammatory diseases, we have recently shown in idiopathic annulo aortic ectasia disease (IAAED) that semicarbazide-sensitive amine oxidase (SSAO) expression in the aneurysmal aortic wall was markedly decreased and correlated with a reduction in elastic lamellar thickness (ELT) [4]. This study did not permit us to determine whether the SSAO reduction represents an early event in aneurysm formation or is only the consequence of arterial injury. Thus elucidation of the contribution of SSAO to arterial function remains an important issue.

SSAO, also called vascular adhesion protein-1, is highly expressed in the plasma membrane of VSMCs of the aortic media, but no specific function for the SSAO enzyme has yet been established [5–7]. Several previous results support the hypothesis that SSAO may be involved in VSMC differentiation, organization of the ECM [8–11], or in regulation of vascular tone [12–14]. Interestingly, SSAO activity is also present in serum and previous studies have shown that SSAO activity is much higher in the human than in the rodent arterial wall [15–17]. Several clinical and experimental investigations have shown that high plasma SSAO activity is observed in arterial diseases. Thus elevated plasma SSAO activity was detected in both type 1 and type 2 diabetes, and is correlated with the severity of retinopathy [18]. An increase in SSAO activity was also observed in chronic heart failure [19], atherosclerosis and obesity [20]. Mouse strains that are more prone to develop atherosclerosis exhibit higher plasma SSAO activity than strains resistant to atherosclerosis [21]. These observations converge to suggest that besides its physiological roles, SSAO could contribute to vascular damage.

It is generally considered that the formation of intra- and extra-molecular cross-links is mediated by lysyl oxidase (LO) [22]. SSAO is known to metabolize primary amines, such as benzylamine, methylamine, or aminoacetone, and to generate the corresponding aldehyde. In view of previous results of studies using molecular modelling [23], it is also conceivable that in addition to soluble primary amines, SSAO may act on aminoacids included in matrix proteins and thus contribute to physiological cross-linking of elastic and collagen fibres. Moreover, a recent study [8] has demonstrated that SSAO-catalyzed deamination of methylamine results in formaldehyde-protein cross-linkage. This observation suggests that endogenous proteins, such as tropoelastin and collagen, may be available for SSAO-mediated cross-link generation.

In view of the above data, the role of amine oxidases in arterial function cannot be limited to LO, but needs to be reevaluated considering a possible involvement of SSAO. Based on pharmacological studies, semicarbazide (SCZ) is the reference compound for inhibiting SSAO activity but in some species, SCZ was reported to inhibit LO activity by up to 60% [24].

In view of the lack of specificity of SCZ, we have chosen to use the recently developed SSAO knockout mouse (SSAO –/–). This model [25] offers a unique opportunity to examine the role of SSAO in arterial mechanical properties and composition of the arterial wall. For the first time, we have thus investigated in detail the vascular phenotype of SSAO –/– mice using high resolution techniques which have been validated in vivo and in vitro. Our first objective was to determine modifications in arterial mechanical properties and vascular structure in SSAO –/– mice. The second objective was to determine the potential alterations in vascular contractile and dilatory functions. In parallel, we have assayed LO activity in an attempt to evaluate whether SSAO invalidation is accompanied by a compensatory increase in LO activity.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Animals
To understand the physiological functions of SSAO, gene targeting techniques were used to disrupt the mouse SSAO/AOC3 gene by replacing a portion of its first exon with a neomycin-resistance (Neor) cassette. Mice homozygous for the null mutation were produced and this mutation was maintained in a pure 129S6 background as previously described by Stolen et al. [25].

Eighteen SSAO –/– mice on the 129S6 genetic background were used in these experiments together with 21 age-matched (22–24 week-old) wild-type mice. All experiments were performed in male animals whereas in vitro mechanical strength of the common carotid artery (CA) was measured in female animals. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2. In vivo arterial mechanical parameters
We simultaneously recorded intra-arterial diameter (left CA) and blood pressure (right CA), in pentobarbital-anesthetized mice as previously described [26,27]. The pressure measurement was made by using a catheter (0.5 cm of PE-10 fused to 3 cm of PE-50; Clay Adams, Parsippany, NJ, USA) connected to a Statham pressure transducer (P23 Db) and a Gould pressure processor. Internal arterial diameter (D) of left CA, 1 cm below the carotid bifurcation, was measured with an ultrasonic echotracking device (NIUS-01, Asulab SA, Neuchâtel, Switzerland). The relationship between the pressure (P) and the lumen cross-sectional area (LCSA) was fitted with the model of Langewouters et al. [28] using an arctangent function and three optimal fit parameters ({alpha}, β, {gamma}) as follows:



Formula

Carotid cross-sectional distensibility (Dist), a derivative of this function, was used to assess the global elastic behaviour of the artery. Circumferential wall stress ({sigma}) and incremental elastic modulus (Einc), which characterizes the intrinsic mechanical properties of the wall material, were calculated with the above-mentioned parameters. Dist, {sigma} and Einc are given by the following equations (according to the general principle of the incompressibility of the arterial wall):



Formula



Formula



Formula

where the media cross-sectional area (MCSA) was determined by histomorphometry.

Since the viscosity of the arterial wall, assessed by the measurement of the energy dissipated at each cardiac cycle, is very low in mice in in vivo conditions [27], it has been ignored in the calculation of elastic parameters.

2.3. Mechanical strength of the carotid artery
The mechanical strength of the intact CA was assessed by determining the in vitro intraluminal pressure required to induce vascular wall rupture [29]. One centimeter of the vessel, free of collateral branches, was carefully dissected. The arterial segment, placed in warm (37 °C) Krebs buffer, was cannulated on a specially designed device and adjusted to its in situ length. An increasing intraluminal static pressure was applied and continuously measured by a pressure transducer (UP4, Pioden, Kent, UK) until rupture of the vascular wall was achieved.

2.4. Histomorphometry
Morphological studies of the arteries were performed on CA fixed in 10% buffered formalin at each animal's mean arterial pressure to provide conditions of fixation close to the physiological in situ state of the vessel. All arterial samples were embedded in paraffin and 6 µm sections were stained with orcein to demonstrate elastic fibres [26]. Arterial thickness and medial cross-sectional area (MCSA) were determined by computer-directed image analysis.

2.5. Isolated thoracic aorta and mesenteric resistance arteries
Vascular contractile and dilatory functions were assessed in isolated thoracic aorta and mesenteric resistance arteries as previously described in mice [30]. Thoracic aortas (2 mm long) and segments of mesenteric arteries (approximately 200 µm external diameter and 2 mm long) were dissected and mounted on a wire-myograph (DMT, Aarhus, DK) as previously described [31]. Briefly, 2 tungsten wires were inserted in the lumen of the arteries and fixed to a force transducer and a micrometer, respectively. Arteries were bathed in a physiological salt solution (PSS) [31] maintained at 37 °C, pH 7.4, 5% CO2 in O2). Arteries were set to the baseline circumference L0 where L0=0.9L100 (i.e. the internal circumference the artery would have in vivo when relaxed and under a transmural pressure of 100 mmHg). The near-maximal active wall tension of the vessel is developed at this circumference [32]. Vessels were allowed to stabilize for 1 h. Artery viability was tested using a potassium-rich solution (80K-PSS).

Isometric tension was recorded and collected by a Biopac data acquisition system (Biopac MP 100, La Jolla, CA, USA) and continuously recorded (Apple computer, Cupertino, CA, USA). Contractions in response to KCl (125 mM) and cumulative concentration–response curves to phenylephrine (PE), serotonin (5HT) and endothelin-1 (ET-1) were obtained. Data was expressed as mN for force development. Concentration–dose response curves for acetylcholine (endothelium-dependent relaxation) and sodium nitroprusside (SNP, endothelium-independent relaxation) were obtained after preconstriction of the artery with phenylephrine (1 nM). Data were expressed as % dilation of phenylephrine-induced preconstriction. EC50 or IC50 (concentration of agonist required to induce the half-maximum response) and Emax (maximal response) were calculated for each artery.

2.6. Biochemical quantification of insoluble elastin and total collagen
Insoluble elastin, total collagen and cell protein contents were measured on descending thoracic aortas as previously described in rats [33]. Briefly, whole aortic segments without homogenization were opened longitudinally and their length measured under a microscope. They were then defatted, dried and their dry weight recorded. Cell proteins were extracted by 0.3% SDS and subsequently assayed and insoluble elastin was purified by the hot alkali method (3x15 min in 0.1 N NaOH, in a boiling water bath) and quantified by weighing. Proteins in the NaOH extract were then hydrolysed, and total collagen was quantified by assaying hydroxyproline in the hydrolysate.

2.7. Enzyme assays
Aortas were taken from 5 wild-type and 5 knockout mice and the surrounding adherent tissue was removed. Aortas were cut in small pieces and homogenized in 1 mM KH2PO4 containing 250 mM sucrose, pH 7.2. These homogenates were used for Western blot and measurement of SSAO activity. For Western blot, SDS-polyacrylamide gel electrophoresis was performed with a mini-protean III apparatus (Bio-Rad). Proteins (20 µg/lane) were mixed with 5x Laemmli's buffer for electrophoresis before loading on an 8% SDS-PAGE acrylamide gel. Proteins were transferred onto a nitrocellulose membrane (Hybond-ECL, Amersham). Blocking was performed in TBS containing 0.1% Tween 20 and 5% of defatted milk powder for 45 min at room temperature. The membrane was incubated for 1 h at room temperature in blocking solution containing 1/1000 primary antibody TK10-79, a rat anti-mouse SSAO antibody [34]. Peroxidase-conjugated goat anti-rat Ig (Dako, Danemark) was used as a secondary antibody (1/2000) in the blocking solution. Nitrocellulose strips were stained using a chemoluminescence detection kit (Amersham).

SSAO activity was assayed by a fluorimetric method (Matsumoto et al., Biochem Pharmacol, 1982). Briefly, 10 µg of protein were pre-incubated for 30 min at 37 °C, in absence or in presence of 500 µM semicarbazide as a SSAO inhibitor. The reaction was started in adding 500 µM benzylamine as a SSAO substrate, 250 µM pargyline (MAO inhibitor), 75 µM Amplex red® (Molecular Probes, Inc.) as a hydrogen peroxide detection probe and 25 UI/ml horseradish peroxidase in 200 µl total volume. H2O2 was detected after 30 min incubation with benzylamine at 545 nm excitation/590 nm emission wavelengths. Results are expressed in nmol H2O2/h/mg of protein.

For measurement of LO activity, the aortas were thawed, dried, weighed and homogenized in 0.15 M NaCl, 16 mM KH2PO4 (pH 8.0), 1 mM phenylmethylsulfonyl fluoride (30 ml/g of tissue). The homogenate was then centrifuged at 12,000 xg for 20 min at 4 °C, and the pellet was resuspended in the same buffer followed by centrifugation as described above. The pellet was resuspended in 4 M urea, 16 mM KH2PO4 (pH 8). Measurement of LO activity was performed as described by [35].

2.8. Statistical analysis
All values are expressed as means±SEM. Unpaired Student's t tests were performed to compare wild type with SSAO –/– mice. For statistical comparison of D–pressure curves, Dist–pressure curves and Einc–wall stress curves between SSAO –/– and +/+ mice, arterial pressure, Dist, and Einc were log transformed to generate linear relationships. The quality of the transformation was checked by calculating the R2 of the linear regression obtained with the new parameters for each individual.

After this transformation, we calculated the mean slopes of these curves as well as the diameter and distensibility at 132 mmHg, a pressure common to both groups, (D132 and Dist132) and the mean wall stress at 1000 kPa of Einc (WS1000). Differences were considered significant at values of p<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. General and haemodynamic parameters
Table 1 shows that the mean body weight of SSAO –/– mice was not significantly different from that of SSAO +/+ animals. There was no change in heart rate or in the mean, systolic, diastolic arterial pressures or pulse pressure in anesthetized animals.


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  		Table 1 General and haemodynamic parameters in SSAO +/+ and SSAO –/– mice

 
3.2. Carotid arterial stiffness
The diameter (D) at MAP of the carotid artery was significantly higher in SSAO –/– mice than in SSAO +/+ animals (Table 2). Carotid wall stress and distensibility calculated at mean arterial pressure (MAP) did not significantly change in SSAO –/– compared to SSAO +/+ mice. The D–pressure, Dist–pressure, Einc/wall stress curves and mechanical strength of the CA of each group are presented on Fig. 1. The D–pressure curve in SSAO –/– mice was shifted upwards and the D132 of SSAO –/– mice was significantly higher than that of SSAO +/+ mice (646±33 and 546±30 µm, p<0.05) and this difference was maintained after adjustment to body weight. The Dist–pressure curve in SSAO –/– mice was shifted downwards compared with that of control mice. The Dist132 of SSAO –/– mice was significantly lower than that of SSAO +/+ mice (4.15±0.60 and 5.77±0.47 1/103 mmHg, p<0.05). The two Einc–wall stress curves were parallel and showed no significant difference in WS1000 between SSAO –/– and +/+ mice (371±20 and 342±29 kPa, NS), indicating no change in arterial stiffness of wall material in SSAO-deficient mice.


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  		Table 2 Mechanical properties and structure of the carotid artery in SSAO +/+ and SSAO –/– mice

 

Figure 1
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  		Fig. 1 Mean carotid artery diameter–AP curves (A), cross-sectional distensibility–AP curves (B), Einc–wall stress curves (C) and mechanical strength (D) in SSAO –/– and SSAO +/+ mice. Panels A, B and C: the diameter–AP curves of SSAO –/– mice were significantly shifted upwards and Dist–AP curves downwards compared with SSAO +/+ animals. The Einc–stress curves were not significantly different between the 2 groups. n=9 for SSAO –/– mice and n=8 for control mice. Panel D: CA rupture pressure was identical in SSAO –/– and +/+ mice. n=6 for SSAO –/– mice and n=7 for control mice.

 
3.3. In vitro mechanical strength of the carotid artery
The in vitro pressure which induced carotid wall rupture was not significantly reduced in SSAO –/– mice compared to SSAO +/+, indicating no change in mechanical strength of the vascular wall (Fig. 1D).

3.4. Morphology of the carotid artery
Table 2 showed that MCSA measured on arteries fixed at 100 mmHg was similar between the two groups. Medial thickness was slightly decreased in SSAO –/– compared to SSAO +/+ (p=0.06), mainly due to the increase in diameter. Histological examination showed that the number, the structure, and the organization of the elastic lamellae in the carotid artery at both low and high magnifications were similar between control and SSAO-deficient mice (Fig. 2A).


Figure 2
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  		Fig. 2 Histomorphometry of the carotid arteries (A) and ECM, insoluble elastin and collagen contents of the aorta (B) in SSAO –/– and SSAO +/+ mice. Panel A: Transverse sections of CAs were stained with orcein to show elastic fibres. Morphology of the elastic lamellae was similar in both genotypes. n=8 control mice and n=9 SSAO –/– mice. Panel B: Dry weight and quantity of ECM proteins, insoluble elastin and total collagen in descending thoracic aorta of control and SSAO –/– mice. All parameters are expressed in mg/cm of the thoracic aorta and elastin and collagen are also expressed as a % of aortic dry weight. n=7 per group. *p<0.05, SSAO –/– mice versus their controls.

 
3.5. Quantification of aortic insoluble elastin and total collagen
We observed a slight but significant increase in aortic dry weight in SSAO-null mice. This was accompanied by a tendency towards an increase in total aortic extracellular proteins and collagen, and a significant increase in insoluble elastin, when expressed as mg/cm (Fig. 2B). After adjustment to body weight, aortic dry weight and elastin content remained significantly higher in SSAO –/– mice. In contrast, when insoluble elastin and total collagen were expressed as a % of aortic dry weight, there was no significant difference between groups, indicating that the proportions of these extracellular fibrous proteins were not altered by the lack of SSAO.

3.6. Pharmacological profile of isolated mesenteric and carotid arteries
Endothelium-dependent (acetylcholine) or -independent (sodium nitroprusside) forms of dilation were not modified in SSAO –/– mice in either thoracic aorta or resistance mesenteric arteries (Table 3, Fig. 3). Similarly, contractions to KCl, phenylephrine, 5HT and ET-1 were not affected by the lack of SSAO (Table 3). These results imply no major smooth muscle or endothelial dysfunction in the absence of SSAO.


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  		Table 3 Contraction to KCL, phenylephrine (PE), serotonin (5HT) and endothelin-1 (ET-1) and dilation to acetylcholine (ACh) and sodium nitroprusside (SNP) in SSAO –/– and SSAO +/+ mice

 

Figure 3
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  		Fig. 3 Vascular response to acetylcholine in SSAO –/– mice. Changes in relaxation in response to acetylcholine in the mesenteric arteries (top) and the thoracic aorta (bottom) isolated from control and SSAO –/– mice. n=5 per group. No significant difference, control versus SSAO –/– mice.

 
3.7. SSAO and LO activities
As expected, SSAO protein and activity were undetectable in aortic tissue of SSAO –/– animals (Fig. 4). In SSAO –/– mice, no difference in aortic LO activity was detectable as compared to control animals, indicating that SSAO invalidation is not accompanied by a compensatory increase in LO activity.


Figure 4
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  		Fig. 4 SSAO, LO activities and Western blot analysis of SSAO expression in SSAO –/– mice. SSAO and LO activities (A–B) were measured in the thoracic aorta (5 rats per group). All activities represent the means±SEM, and are expressed in nmol/h/mg of proteins. SSAO (97 kDa) was detected using a rat anti-mouse SSAO antibody. SSAO activity and protein were undetectable in aortic tissue of SSAO –/– animals. In SSAO –/– mice, no change in aortic LO activity was detectable as compared to control animals.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
This is the first study of arterial function in relation to the invalidation of SSAO in mice. This model is well adapted to analyze the exact role of SSAO in the vascular wall, since no highly SSAO-selective inhibitor is currently available. SSAO –/– mice do not express SSAO in their arterial walls. Our results show that SSAO-deficient mice present an increase in arterial diameter whereas arterial mechanical properties and endothelial and smooth muscle function were normal. They also suggest that elastin cross-linking processes were efficient in arteries from SSAO –/– mice, as the insoluble elastin content was not decreased compared to controls.

The first hypothesis to be tested was that SSAO might be involved in elastin cross-linking processes during vessel development. In this context, we have previously shown that in IAAED, SSAO protein levels were markedly decreased and correlated with a reduction in elastic lamellar thickness [4] suggesting its implication in arterial diseases. Langford et al. [9] have shown that pharmacological inhibition of SSAO in a growing rat model led to striking elastic fibre disorganization, possibly due to reduced cross-linking of elastin monomers. Moreover, it has been reported that in transgenic mice overexpressing SSAO in VSMCs, an abnormal structure of the aortic elastic lamellae was exhibited, characterized by stretching and unfolding of elastic fibres [11]. Despite these reports, the present results do not support the hypothesis of a contribution of SSAO to elastin cross-linking.

The second hypothesis was that SSAO might participate in the general process of smooth muscle development and regulation of vascular tone. We have previously reported that SSAO expression and activity were dependent upon the degree of cell differentiation, thus raising the possibility that changes in SSAO activity could be linked to variations in VSMC phenotype [36]. Alternatively, it has been suggested that SSAO could influence arterial vascular tone, although contradictory results have been reported [12–14]. Vidrio et al. [12] have proposed that SSAO-mediated hydrogen peroxide production could increase vascular tone, and enhance hydralazine-induced vasodilation. Our present results in vitro do not support a role for SSAO in vascular contractile and dilatory function, as these were normal in the SSAO –/– mouse, implying an efficient control of vascular tone in this model. Interestingly, Conklin et al. have recently suggested that in isolated human arteries, SSAO activation by methylamine mediated a vasorelaxant action [14]. Thus, it would be of interest to examine whether SSAO –/– mice retain or not a vasodilatory response to administration of SSAO substrates. However, it should be pointed out that the concentration of methylamine required to provoke this response in human arteries was supraphysiological (in the mM range).

The Einc/wall stress curve of SSAO –/– mice was similar to that of control mice, indicating that the intrinsic stiffness of wall components in SSAO –/– mice was not modified. This result is in agreement with the lack of difference between the two groups both for the relative content of ECM proteins (expressed as a percentage) and for vascular smooth muscle and endothelial function, two major determinants of arterial stiffness [37,38]. The difference in arterial distensibility, which integrates both arterial geometry and mechanical properties, may be explained by the difference in arterial diameter between the two groups.

SSAO –/– mice showed no reduction in the rupture pressure of the carotid wall compared to control mice, indicating that the mechanical strength of the vascular wall is not modified. Collagen cross-linking is generally considered to be responsible for mechanical strength of tissues at high stress levels [22,39], but insoluble collagen was not measured here and so we cannot directly evaluate collagen cross-linking. However, since our histological and biochemical results suggest that elastin cross-linking was not decreased and in view of the unchanged carotid rupture pressure, it is very unlikely that collagen fibres were affected.

Since SSAO –/– mice had normal elastin cross-linking as assessed by insoluble elastin content, and normal vascular contractile and dilatory functions in vitro, the increase in luminal arterial diameter at similar blood pressure levels must be interpreted cautiously. The slight reduction in medial thickness in SSAO –/– compared to SSAO +/+ is mainly due to the increase in diameter with similar MCSA and this does not support the hypothesis of arterial wall thinning. The increased diameter in the KO mice may be related to the tendency towards an increase in circumferential wall stress calculated at MAP, although this difference was not statistically significant. Alternatively, it may be related to smooth muscle relaxation, but the responses of small muscular arteries to various agonists did not differ between the two groups. In particular, we did not detect any modification in acetylcholine-induced vasorelaxation, rendering any major endothelial dysfunction unlikely. Finally, we found no difference between the two groups concerning elastin cross-linking (this result may also be extrapolated to collagen cross-linking), which could explain a difference in diameter. However, the increase in large arterial diameter associated with a tendency towards a general increase in arterial constituents per cm may suggest some degree of expansive remodeling increased arterial caliber, raising the possibility that SSAO exerts a role during vascular development and growth via other mechanisms. During this phase, blood flow plays an important role in determining vascular growth [40], via the shear stress exerted by blood flow on the endothelium. However, the endothelial response to flow was not tested here as it was not one of the original aims of our study.

Finally, the invalidation of SSAO in mice does not induce arterial aneurysm formation or rupture of the vascular wall, as previously described for LO [41]. The finding of a general increase in arterial diameter in mice lacking SSAO for a given level of arterial pressure does not support a role for this enzyme in focal aneurysm formation in IAAED patients although SSAO was focally decreased in this disease [4]. Although the mouse model of invalidation should be interpreted in the context of development and growth, while human aneurysmal disease occurs later in life, the present findings suggest that SSAO is not causal in IAAED. This model is also characterized by the absence of any increase in arterial LO activity. This finding suggests that no functional interaction between SSAO and lysyl oxidase exists within the vascular wall, although their ECM substrates are potentially common. Our results differ from previously published data regarding the involvement of SSAO in elastin cross-linking processes and the induction of arterial damage by pharmacological inhibition of SSAO. The differences observed with SCZ treatment may be related to the lack of specificity of SCZ, which largely inhibits LO, as previously described. Determination of the exact contribution of these two amine oxidases to ECM formation and tissue development requires complementary investigations at various ages, especially in older animals.

In conclusion, we have shown that invalidation of SSAO in mice modifies arterial geometry but does not influence mechanical properties. Although mice lacking SSAO present a higher carotid arterial diameter at similar blood pressure levels, our results do not support a major role for SSAO in the organization of the elastic fibre network, elastin cross-linking processes and in vascular endothelial or smooth muscle function. However, since SSAO activity is much higher in the human than in the rodent arterial wall [15–17], defining the exact implication of SSAO in physiology and pathology of human arteries remains a major challenge.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
We acknowledge Pr Michel Safar for his helpful discussion of the manuscript. N. Mercier is a recipient of a Marie Curie fellowship grant. This study was supported by grants from the Academia of Finland, INSERM, CNRS, ANR, Universities of Nancy (Université Henri Poincaré), Paris 6 and Paris 11.


   Notes
 
Time for primary review 29 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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