Semicarbazide-sensitive Amine Oxidase in Annulo-aortic Ectasia Disease : Relation to Elastic Lamellae-associated Proteins
Inserm U441 (IS,DL,DD,JB,J-MDL), Pessac, France, and CNRS UMR 7079 (KEH,NM,BF) and Inserm EMI U01-07 (CL,PL), Paris, France
Correspondence to: Jean-Marie Daniel Lamazière, Inserm U441, Université Victor Segalen Bordeaux 2, avenue du Haut Lévêque, 33600 Pessac, France. E-mail: jean-marie.d-lamaziere{at}bordeaux.inserm.fr
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: amine oxidase aneurysm idiopathic annulo-aortic ectasia disease elastic fiber
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aneurysms may result from destruction of the extracellular matrix (ECM) via inflammatory proteases, such as those observed in complicated atherosclerosis (Fontaine et al. 2002), or from an alteration in elastic fiber organization, which occurs in cases of quantitative and/or qualitative defects of some molecular components of elastic or collagen fibers, the major ECM constituents of the arterial wall. However, a genetically determined deficit in elastin leads to VSMC proliferation during development and arterial stenosis (Li et al. 1997
,1998
). By contrast, Marfan syndrome, a genetic disorder caused by mutations in the gene for fibrillin-1, a component of the microfibrils, is accompanied by aortic aneurysms due to a lack of elastic fiber integrity (Dietz et al. 1991
; Kainulainen and Peltonen 1991
). Some thoracic aortic aneurysms associated with aortic insufficiency (aortic regurgitation as defined by Doppler echocardiography), which fit into none of the above-mentioned categories, are classified as idiopathic annulo-aortic ectasia disease (IAAED) and remain pathologically undefined (Savunen and Aho 1985
; Savunen 1986
). Because the etiology of IAAED is not related to any known genetic disorder, it is an interesting model in which to study possible, as yet undiscovered, intrinsic structural disorders of the ECM.
During embryonic arterial development and growth, functional elastic fibers are formed by cross-linking of tropoelastin. The first step in this process is the formation of -aldehyde, allysine, through oxidation of
-amino groups by the secreted copper-dependent enzyme lysyl-oxidase (Lox) (Bedell-Hogan et al. 1993
; Li et al. 2000
; Kagan and Li 2003
). Subsequent non-enzymatic condensation of modified and unmodified lysines leads to formation of characteristic bifunctional and tetrafunctional cross-links specific to insoluble elastin (Smith-Mungo and Kagan 1998
; Maki et al. 2002
; Kagan and Li 2003
). Assembly of collagen molecules into fibrils also requires the formation of cross-links by Lox. Therefore, the process of cross-linking is essential for correct elastic and collagen fiber function. In humans, the importance of the cross-linking is illustrated by the Menkes syndrome, a functional deficit in Lox related to a mutation in the gene coding for a copper-transporting ATPase, that results in a fragmentation of elastic fibers (Smith-Mungo and Kagan 1998
).
Semicarbazide-sensitive amine oxidase (SSAO), also called vascular adhesion protein-1 (VAP-1), is another copper-containing amine oxidase. A high activity of SSAO is associated with VSMCs localized in the media of the mammalian aorta (Lyles 1996; Jalkanen and Salmi 2001
), and its expression is dependent on the level of maturation of VSMCs (El Hadri et al. 2002
). Using a pharmacological approach, Langford et al. (1999)
have shown that chronic blockade of vascular SSAO activity leads to a striking disorganization of the elastic fiber architecture within the aortic media, accompanied by a decrease in the mature elastin content and an increase in collagen, leading to aortic dilatation (Langford et al. 1999
). Therefore, it is conceivable that, like Lox, SSAO may contribute to the cross-linking processes that participate in the organization of elastin and collagens. Very recently, it has been reported (Göktürk et al. 2003
) that in a murine transgenic model, overexpression of human SSAO in smooth cells leads to abnormal structure of the aortic elastic laminae. These studies have suggested that like Lox, SSAO may contribute to the organization of the elastin network. SSAO has been shown to be expressed by human adult aortic VSMCs but is not implicated in atherosclerotic or inflammatory diseases (Jaakkola et al. 1999
). To our knowledge, no study has evaluated SSAO expression in human aneurysmal disease.
The aim of our study was to examine the expression of several proteins that could be involved in elastic fiber organization at the aneurysmal site in IAAED. These molecules were analyzed in parallel with elastic lamellar thickness (ELT) considered as an index of elastic fiber structural integrity. Expression of the amine oxidases (Lox and SSAO) was evaluated by immunohistochemistry (IHC) and confocal microscopy. We also investigated alterations in SMC differentiation using monoclonal antibodies (MAbs) specific to sm-myosin heavy-chain (sm-MHC) and sm--actin. We observed that although SSAO and Lox were easily detectable in the vicinity of elastic fibers of normal aorta, only SSAO expression was dramatically reduced in the media of IAAED, even after adjustment for decreased elastic fiber content. SSAO content was positively correlated with ELT in both control and IAAED subjects. Whether this marked decrease in SSAO expression in IAAED is simply the consequence of elastic fiber alterations and SMC dedifferentiation, or whether it itself contributes to the disorganization of elastic lamellae, cannot be concluded from this study and requires further investigation.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Normal aortic tissues were obtained from patients who had died in accidents and who were used as heart graft donors, in accordance with the requirements of the local Ethical Committee (CHU Bordeaux). All the controls were sex- and age-matched and free of coronary artery disease and of major vascular risk factors including diabetes, hypercholesterolemia, hypertension, and smoking. This study was performed in a clinical-research hospital project (PHRC-CHU, Bordeaux, France).
Histomorphometry, Immunohistochemistry, and Confocal Microscopy
Aortic samples were obtained during Bentall's surgery. Tissues were placed in Dulbecco's modified Eagle's medium (Life Technologies EUROBIO, Courtaboeuf, France). Samples were then separated into two fragments, one fixed in paraformaldehyde for histochemical studies and one freeze dried for immunohistology. Aortic sections already paraformaldehyde-fixed and paraffin-embedded were stained with Weigert's resorcin/fuschin and Masson's trichrome for elastin fiber, collagen content, and SMC identification (Michel et al. 1994; Sauvage et al. 1997
). Thickness of elastic lamellae was measured across the entire media on 10 different fields of each Weigert's-stained section to take into account all elastic lamellae of an aneurysmal area.
MAbs specific to -sm-actin, sm-MHC, collagen type IV, and elastin were purchased from Sigma (St Louis, MO). Antibodies to Lox were a gift of Dr P. Sommer (IPCB CNRS; Lyon, France), while antibodies to metalloproteinase (MMP)-2 and to MMP-9 were from Chemicon (Temecula, CA) and from Santa Cruz Technology (Santa Cruz, CA), respectively. SSAO antibody was a kind gift of Dr S. Jalkanen (Medicity Research Laboratory, Department of Biochemistry and Pharmacy; Turku University, Finland).
Because antigenic preservation is sensitive to tissue fixation conditions, tissues were quickly deep frozen in liquid nitrogen, then freeze dried and embedded in paraffin as described previously (Louis et al. 2000). Bound primary antibodies were detected using biotinylated anti-mouse, anti-rat, anti-rabbit, or anti-goat secondary antibodies and the streptavidin/biotinylated horseradish peroxidase complex (Amersham Pharmacia Biotech; Orsay, France). The final complex was visualized using a DAB/peroxidase kit from Vector (Burlingame, CA). Sections were counterstained in Mayer's hemalun, dehydrated, and mounted in a EUKITT medium (Kindler; Freiburg, Germany). Controls were performed by omitting the primary antibody.
Histomorphometric data and immunostaining were quantified at high-power magnification by color video image analysis using an IBM PC (Daniel Lamazière et al. 1993). The video camera was connected to a microscope. The software Quancoul (Quant'Image; INSERM U441, Pessac, France) defined true colors on the basis of three independent parameters (hue, intensity, and saturation). The parameters were calibrated against a background of control antigen as previously described (Daniel Lamazière et al. 1993
; Bézie et al. 1998
). To account for the color intensity as an index of the amount of antigenic detection, we expressed the collagen density and antigenic content in arbitrary units (AU) as the product of the positively labeled surface by the hue intensity and optical density of the same positive pixels (Daniel Lamazière et al. 1993
; Bézie et al. 1998
). At least 10 sections for each aorta were measured to calculate the mean value for each sample. Because the surgical arterial biopsies did not allow study of the full thickness layer, all measurements were normalized to normal or injured arterial area. We controlled the validity of our quantitative immunological method in accordance with ELISA quantification (Figure 1)
.
|
Statistical Analysis
Results are expressed as means ± SD. Statistical comparisons between groups were made using the non-parametric Mann-Whitney test. Clinical parameters were analyzed after adjustment for age and sex. Differences in SSAO expression between IAAED and controls were analyzed after adjustment for elastin content using a general linear model. Univariate correlations and multiple regression analysis with stepwise selection were used to assess the determinants of ELT and clinical IAAED parameters. Statistical analysis was carried out using the NCSS 2000 software. p<0.05 was considered statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
Regression Analysis
Figure 6
shows that ELT was positively correlated with SSAO content in both the IAAED group (R2 = 0.81; p<0.0001) and in controls (R2 = 0.48; p<0.03). There was no significant correlation between ELT and Lox in either the IAAED group or controls. There was no significant correlation between sm--actin and sm-MHC content and the amount of SSAO in either group, or between ELT and sm-MHC content.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The decrease in SSAO expression in IAAED patients and its positive correlation with ELT should be interpreted with caution. One must bear in mind that our study was performed on IAAED specimens that were probably at a late stage of the evolution of the disease. It is therefore impossible to determine whether during the natural history of IAAED the SSAO decrease represents an early event that precedes the elastic fiber disorganization. Because elastin turnover is very slow (Brown-Augsburger et al. 1996), this hypothesis can be considered only if SSAO contributes in some way to the postnatal stabilization of the elastin network.
Several lines of evidence in our experimental data and those of others suggest that SSAO may be involved in elastic fiber organization: (a) the presence of a positive SSAO gradient in IAAED patients from the most- to the least-affected areas. Interestingly, we observed in the transition zone (see Figure 2, zone 2) a marked decrease in SSAO immunostaining, whereas ELT was only slightly decreased, suggesting that SSAO downregulation may precede ELT decrease and disorganization. (b) The significant correlation between ELT and SSAO expression observed in IAAED was maintained in control vessels. In addition, when a multiple regression analysis was performed, the SSAO:elastin ratio correlated significantly with ELT compared with other factors tested in the model. (c) Langford et al. (1999) have shown that SSAO inhibition in a growing rat model led to striking elastic fiber disorganization, possibly due to a lack of cross-linking of elastin monomers. Moreover, Göktürk et al. (2003)
have recently reported an abnormal structure of the aortic elastic laminae characterized by a higher ELT in transgenic mice overexpressing SSAO in SMCs. However, these findings, taken together, although suggestive, do not demonstrate that SSAO plays a role throughout life in the stabilization of the elastin network.
At a biochemical level, mechanisms by which SSAO might contribute to the organization of the ECM network remain speculative. Lox, which is structurally related to SSAO, is involved in collagen and elastin cross-linking and in chemotaxis of VSMCs (Kagan et al. 1984; Smith-Mungo and Kagan 1998
; Li et al. 2000
; Kagan and Li 2003
). This amine oxidase can use NH2 in lysine side chains in proteins as a substrate. SSAO is generally considered to metabolize primary amines, such as benzylamine, methylamine, or aminoacetone, to generate the corresponding aldehyde, hydrogen peroxide, and ammonia. Interestingly, a recent study (Salmi et al. 2001
) has shown by molecular modeling that a polypeptide can fit into a groove on the surface of SSAO at a position that overlies the catalytic center of SSAO. Overall, in endothelial cells, a lysine-containing peptide (GGGGKGGGG) fitting into the groove decreases SSAO-mediated hydrogen peroxide production in response to benzylamine, indicating that it interferes with enzyme catalytic activity (Salmi et al. 2001
). By contrast, irrelevant lysine-containing peptides have no effect on SSAO activity. It is therefore conceivable that in addition to soluble primary amines, Lox and SSAO may act on amino acids included in matrix proteins. In addition, considering our methodological approach and the size of arterial samples, it is not possible to establish the relationship between SSAO tissue expression and activity, but this has previously investigated in the work of Jaakkola et al. (1999)
and Andres et al. (2001)
. In this context, further morphological, biochemical, and molecular investigations will be required to determine this relationship and the exact nature of the SSAO substrate(s) in the blood vessels.
However, the advanced state of disorganization of the arterial wall observed in IAAED does not allow us to establish this potential role of SSAO. Indeed, SMC dedifferentiation and elastin fragmentation and degradation could themselves account for the marked reduction in SSAO expression. Our results show that in contrast to atheroslerotic lesions, there is no increase in MMP2 and MMP9 expression in IAAED, suggesting that they do not play a role in elastolysis in this disease. However, we cannot exclude a modification in MMP activities as previously studied in metastatic rat liver using zymography and IHC (Mook et al. 2003). Although the relationship between MMP expression and activity has been well documented in atherosclerotic aneurysms, this relationship has never been explored in IAAED. In addition, other elastolytic enzymes exist, some of which can be produced by the VSMCs. In this context, the decrease in sm-MHC immunolabeling demonstrated that the differentiation process of VSMCs is altered in IAAED. This result is in agreement with our study in vitro (El Hadri et al. 2002
) showing that rat VSMCs in the undifferentiated state express low levels of SSAO, whereas VSMC differentiation is accompanied by a large increase in SSAO gene and protein expression. Alternatively, recent studies have shown that elastin plays a direct role in controlling the VSMC phenotype. Therefore, it is conceivable that the reduction in elastin promotes VSMC dedifferentiation, thus contributing to SSAO downregulation in the vascular wall.
In contrast to SSAO, we observed an increase in Lox expression in IAAED, but Lox expression did not correlate with ELT in the controls or the IAAED population. However, using multiple regression analysis on the whole population, a decreased Lox:elastin ratio appeared as a significant determinant of ELT, but with a lower contribution than the increased SSAO:elastin ratio. In light of the importance of Lox in the biogenesis of the ECM by cross-linking both collagen and elastin (Smith-Mungo and Kagan 1998), we cannot exclude the possibility that this increase in Lox expression in IAAED represents an adaptive mechanism in response to ECM disorganization. Whether a cross-talk mechanism exists between Lox and SSAO expression and/or function in the arterial wall remains an open question.
In conclusion, the present study demonstrates that SSAO is expressed along elastic lamellae in intact arterial wall. In IAAED, SSAO, in contrast to Lox, was markedly decreased, associated with severe disorganization of the elastic lamellar network. The relationship between SSAO expression and ELT suggests that this amine oxidase may be involved in elastic fiber organization. However, in advanced IAAED, the deficit in SSAO expression could be secondary to elastic fiber fragmentation and reduction and/or to VSMC dedifferentiation. Experiments using SSAO gain or loss of function will be required to establish the potential interaction between SSAO and elastic fibers.
![]() |
Acknowledgments |
---|
We thank Michel Safar and Mary Osborne-Pellegrin for helpful discussion.
![]() |
Footnotes |
---|
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andres N, Lizcano JM, Rodriguez MJ, Romera M, Unzeta M, Mahy N (2001) Tissue activity and cellular localization of human semicarbazide-sensitive amine oxidase. J Histochem Cytochem 49:209217
Bedell-Hogan D, Trackman P, Abrams W, Rosenbloom J, Kagan H (1993) Oxidation, cross-linking, and insolubilization of recombinant tropoelastin by purified lysyl oxidase. J Biol Chem 268:1034510350
Beighton P, de Paepe A, Danks D, Finidori G, Gedde-Dahl T, Goodman R, Hall JG, et al.l (1988) International nosology of heritable disorders of connective tissue, Berlin, 1986. Am J Med Genet 29:581594[Medline]
Bézie Y, Daniel Lamazière J-M, Laurent S, Challande P, Cunha RS, Bonnet J, Lacolley P (1998) Fibronectin expression and aortic wall elastic modulus in spontaneously hypertensive rats. Arterioscler Thromb Vasc Biol 18:10271034
Brown-Augsburger P, Broekelmann T, Rosenbloom J, Mecham RP (1996) Functional domains on elastin and microfibril-associated glycoprotein involved in elastic fibre assembly. Biochem J 318:149155[Medline]
Clark JM, Glagov S (1985) Transmural organization of the arterial media. The lamellar unit revisited. Arteriosclerosis 5:1934[Abstract]
Daniel Lamazière JM, Lavallée J, Zunino C, Larrue J (1993) Semiquantitative study of the distribution of two cellular antigens by computer-directed color analysis. Lab Invest 68:248252[Medline]
Dietz HC, Cutting GR, Pyeritz RE, Maslen CL, Sakai LY, Corson GM, Puffenberger EG, et al. (1991) Marfan syndrome caused by a recurrent de novo missense mutation in the fibrillin gene. Nature 352:337339[CrossRef][Medline]
El Hadri K, Moldes M, Mercier N, Andreani M, Pairault J, Feve B (2002) Semicarbazide-sensitive amine oxidase in vascular smooth muscle cells: differentiation-dependent expression and role in glucose uptake. Arterioscler Thromb Vasc Biol 22:8994
Fontaine V, Jacob MP, Houard X, Rossignol P, Plissonnier D, Angles-Cano E, Michel JB (2002) Involvement of the mural thrombus as a site of protease release and activation in human aortic aneurysms. Am J Pathol 161:17011710
Göktürk C, Nilsson J, Nordquist J, Kristensson M, Svensson K, Soderberg C, Israelson M, et al. (2003) Overexpression of semicarbazide-sensitive amine oxidase in smooth muscle cells leads to an abnormal structure of the aortic elastic laminas. Am J Pathol 163:19211928
Jaakkola K, Kaunismaki K, Tohka S, Yegutkin G, Vanttinen E, Havia T, Pelliniemi LJ, et al. (1999) Human vascular adhesion protein-1 in smooth muscle cells. Am J Pathol 155:19531965
Jalkanen S, Salmi M (2001) Cell surface monoamine oxidases: enzymes in search of a function. Embo J 20:38933901
Kagan HM, Li W (2003) Lysyl oxidase: properties, specificity, and biological roles inside and outside of the cell. J Cell Biochem 88:660672[CrossRef][Medline]
Kagan HM, Williams MA, Williamson PR, Anderson JM (1984) Influence of sequence and charge on the specificity of lysyl oxidase toward protein and synthetic peptide substrates. J Biol Chem 259:1120311207
Kainulainen K, Peltonen L (1991) Marfan gene discovered. Ann Med 23:395396[Medline]
Langford SD, Trent MB, Balakumaran A, Boor PJ (1999) Developmental vasculotoxicity associated with inhibition of semicarbazide-sensitive amine oxidase. Toxicol Appl Pharmacol 155:237244[CrossRef][Medline]
Li DY, Brooke B, Davis EC, Mecham RP, Sorensen LK, Boak BB, Eichwald E, et al. (1998) Elastin is an essential determinant of arterial morphogenesis. Nature 393:276280[CrossRef][Medline]
Li DY, Toland AE, Boak BB, Atkinson DL, Ensing GJ, Morris CA, Keating MT (1997) Elastin point mutations cause an obstructive vascular disease, supravalvular aortic stenosis. Hum Mol Genet 6:10211028
Li W, Liu G, Chou IN, Kagan HM (2000) Hydrogen peroxide-mediated, lysyl oxidase-dependent chemotaxis of vascular smooth muscle cells. J Cell Biochem 78:550557[CrossRef][Medline]
Louis H, Lavie J, Lacolley P, Daret D, Bonnet J, Daniel Lamazière JM (2000) Freeze-drying allows double non radioactive ISH and antigenic labelling. J Histochem Cytochem 48:499508
Lyles GA (1996) Mammalian plasma and tissue-bound semicarbazide-sensitive amine oxidases: biochemical, pharmacological and toxicological aspects. Int J Biochem Cell Biol 28:259274[CrossRef][Medline]
Maki JM, Rasanen J, Tikkanen H, Sormunen R, Makikallio K, Kivirikko KI, Soininen R (2002) Inactivation of the lysyl oxidase gene Lox leads to aortic aneurysms, cardiovascular dysfunction, and perinatal death in mice. Circulation 106:25032509
Michel JB, Heudes D, Michel O, Poitevin P, Philippe M, Scalbert E, Corman B, et al. (1994) Effect of chronic ANG I-converting enzyme inhibition on aging processes. II. Large arteries. Am J Physiol 267:R124135[Medline]
Mook OR, Van Overbeek C, Ackema EG, Van Maldegem F, Frederiks WM (2003) In situ localization of gelatinolytic activity in the extracellular matrix of metastases of colon cancer in rat liver using quenched fluorogenic DQ-gelatin. J Histochem Cytochem 51:821829
Salmi M, Yegutkin GG, Lehvonen R, Koskinen K, Salminen T, Jalkanen S (2001) A cell surface amine oxidase directly controls lymphocyte migration. Immunity 14:265276[Medline]
Sauvage M, Jacob MP, Osborne-Pellegrin M (1997) Aortic elastin and collagen content and synthesis in two strains of rats with different susceptibilities to rupture of the internal elastic lamina. J Vasc Res 34:126136[CrossRef][Medline]
Savunen T (1986) Annulo-aortic ectasia. A clinical, structural and biochemical study. Scand J Thorac Cardiovasc Surg Suppl 37:145[Medline]
Savunen T, Aho HJ (1985) Annulo-aortic ectasia. Light and electron microscopic changes in aortic media. Virchows Arch A Pathol Anat Histopathol 407:279288[Medline]
Smith-Mungo LI, Kagan HM (1998) Lysyl oxidase: properties, regulation and multiple functions in biology. Matrix Biol 16:387398[CrossRef][Medline]