1INSERM U 460, Bâtiment 13, Hôpital Bichat-Claude Bernard, 46 rue Henri Huchard, 75877 Paris Cedex 18, France
2Department of Cardiology, Hôpital Bichat-Claude Bernard, 46 rue Henri Huchard, 75877 Paris Cedex 18, France
3Department of Pathology, Hôpital Bichat-Claude Bernard, 46 rue Henri Huchard, 75877 Paris Cedex 18, France
4Banque de Tissus Humains, Hôpital Saint-Louis, 1 avenue Claude Vellefaux 75475, Paris Cedex 10, France
5Department of Cardiac Surgery, Hôpital Bichat-Claude Bernard, 46 rue Henri Huchard, 75877 Paris Cedex 18, France
Received 1 October 2004; revised 10 February 2005; accepted 3 March 2005; online publish-ahead-of-print 12 April 2005.
* Corresponding author. Tel: +33 1 40 25 86 17; fax: +33 1 40 25 86 02. E-mail address: jacob{at}bichat.inserm.fr
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Abstract |
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Methods and results Histological analysis of pathological valves [aortic stenosis (AS) (n=49), aortic regurgitation (AR) (n=23)] and control valves (n=8) was performed. The main tissue abnormalities (calcification, inflammatory cells, and capillaries) observed in AS were less severe or absent in AR. However, both groups of pathological valves displayed similar histological signs of extracellular matrix (ECM) remodelling. Biochemical analysis of MMPs and TIMPs (gelatin and casein zymography and ELISA) was performed on valve extracts. MMP-2 activity was not significantly different in control and pathological valves. Increases in MMP-9 and MMP-3 in AS demonstrated an inflammatory state. Finally, there was a four- to seven-fold increase of TIMP-1 in pathological valves. TIMP-1, TIMP-2, and MMP-2 were synthesized by the valvular interstitial cells in primary culture.
Conclusion This study demonstrates the involvement of the MMP/TIMP system in ECM remodelling of both AS and AR. These findings provide evidence of inflammatory injury more severe in AS than in AR and involvement of mesenchymal cell response.
Key Words: Aortic stenosis Aortic regurgitation Matrix metalloproteinases Tissue inhibitor of matrix metalloproteinases Valves
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Introduction |
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Abnormal aortic valve function observed in AS and AR likely results from tissue remodelling, involving especially the extracellular matrix (ECM). The processes involved in the abnormal ECM accumulation or destruction are uncertain. In physiological conditions, the ECM of any tissue is maintained by a rigorously controlled balance between the synthesis and the breakdown of its component proteins. A disequilibrium between the synthesis of ECM components and their degradation leads to a pathological remodelling of the ECM.4 Matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of MMPs (TIMPs), play central role in the degradation process. Recent findings suggest that inflammation contributes to valve diseases.3 The link between inflammation and remodelling has been established in atherosclerosis, but has not been clearly established in valve disease.4
Our hypothesis is that the ECM remodelling and the associated systems of degradation, especially the MMPs, play a major role in pathological valvular remodelling observed in AS and AR. MMP-2 is the main MMP secreted by mesenchymal cells, whereas MMP-3, MMP-9, and, to a lesser extent, MMP-7 are synthesized by inflammatory cells.4
Our study, based on a histological and biochemical approach, evaluated the MMP/TIMP balance using AR and AS valves from patients who required surgery for severe aortic dysfunction. These pathological valves were compared with control valves obtained at the bank of human tissues. In addition, interstitial cells from pathological valves were cultured to analyse the contribution of these cells to the secretion of MMPs and TIMPs.
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Methods |
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Histological analysis
After fixation in a 4% buffered formaldehyde solution and embedding in paraffin, 5 µm sections were stained for histological examination using haematoxylinphloxinsaffron, Masson's trichrome, orcein, and Alcian Blue. Pathological findings (calcification, neovascularization, inflammatory infiltrates, etc.) and ECM alterations [disorganization of collagen bundles, increased loose connective tissue (myxoid tissue), fragmentation or stratification of elastic fibres] were evaluated using a semi-quantitative analysis performed by two independant investigators without knowledge of the origin of the tissues. For this purpose, three sections of each valve were observed at low magnification and divided into four to five fields. Each parameter was graded 03 per field and expressed as the mean value per valve.
Biochemical analysis
Protein extraction
Valve leaflet fragments were homogenized in a 2 mol/L guanidinium chloride solution containing 0.01 mol/L CaCl2, 0.2% Triton X-100, and 0.05 mol/L TrisHCl, pH 7.5 (3 mL/g wet weight) using a Polytron® as previously described.5 After centrifugation (10 000 g, 25 min, and 4°C), supernatants were dialyzed at 4°C during 48 h (Spectrapor membrane 68000 MWC). The dialyzed extracts were centrifuged (10 000 g, 25 min, and 4°C) and protein concentration was determined by the Bradford assay (Bio-Rad).6
Gelatin and -casein zymography
MMP-2 and MMP-9 activities were measured using gelatin zymography and MMP-3 and MMP-7 using casein zymography. Valve extracts containing 10 µg (gelatin zymography) or 30 µg (casein zymography) of proteins were analysed by electrophoresis on 10% sodium dodecyl sulphatepolyacrylamide gels (SDSPAGE) containing 1 mg/mL gelatin (Sigma) or -casein (Fluka) under non-reducing conditions as previously described.5 Gelatin gels were incubated at 37°C for 19 h, whereas casein gels were incubated for 48 h. At the end of the incubation, gels were stained in a Coomassie Blue solution during 1 h, then transferred to a 10% acetic acid30% ethanol solution for 1 h and kept in 10% acetic acid solution. Enzyme activities were quantified by densitometry using the NIH Image 1.60 ppc software. Further characterization of enzyme activities was performed by establishing their inhibition spectra (addition of 30 mmol/L EDTA or 3 mmol/L Pefabloc) and by using western blot. For western blotting, electrophoresis was performed as described by Laemmli on 10% SDSPAGE gels under reducing conditions.5 Antibodies to MMP-2, -3, -7, and -9 (Chemicon) were used at the concentration of 1 g/mL and horseradish peroxidase IgG (Dako) was used after dilution (1/1000). Peroxidase activity was detected using a chemoluminescence reagent (NEN, Perkin Elmer).
ELISA
TIMP-1 and TIMP-2 levels in valve extracts were measured using an ELISA assay (Amersham). The analysis of correlations between the TIMP-1 and the TIMP-2 concentrations and their inhibitory capacities visualized on reverse gelatin zymography,7 was performed using 20 valve extracts.
Cell culture
Interstitial cells, obtained by the explant method, were cultured in smooth muscle basal medium 2 (Promocell®) supplemented with 10% fetal calf serum. Immunohistochemical analysis using antibodies against vimentin, smooth muscle -actin, desmin, smooth muscle-myosin, and prolyl-4-hydroxylase was performed on cells seeded in Labtek wells and fixed in 3.7% paraformaldehyde. After two or three passages, cells were seeded in 1.9 cm2 wells and cultured to confluency. After 48 h in serum-free medium (deprivation), cells were further incubated in serum-free medium in the absence or in the presence of tumour necrosis factor (TNF)-
and interleukin (IL)-1ß (2 and 20 ng/mL). At the end of an additional 48 h incubation, culture media were collected, cells were rinsed with PBS and lysed in TrisHCl 0.05 mol/L, Triton 0.1%, and EDTA 0.01 mol/L, pH 7.5. After the measurement of protein concentration, MMP activities were analysed using gelatin or casein zymography and TIMP concentrations by ELISA in cell culture media and cell extracts.
Statistical analysis
Data were expressed as median values (25th75th percentile) unless stated. Comparison between the three groups of valves was performed using the non-parametric KruskallWallis analysis of variance. Comparisons between subgroups used the MannWhitney U test with the Bonferroni correction for multiple comparisons. Correlations were analysed using the Spearman's test. All tests were two-sided and differences were considered significant when P<0.05.
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Results |
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Biochemical analysis
MMP activity
The activity of the gelatinase MMP-2 was not different between control and pathological valves (P=0.42), whereas MMP-9 activity was significantly different between the three groups (P=0.005) (Figure 2C and D). MMP-9 activity was higher in AS than in AR. MMP-9/MMP-2 ratio differed between the three groups (P=0.007). It was significantly increased in AS compared with AR (Figure 2E). This MMP-9/MMP-2 ratio was higher in AS than in controls, the difference being of borderline significance.
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TIMP concentration
TIMP-2 and TIMP-1 were detectable in both normal and pathological aortic valves. TIMP-2 concentration did not differ among the three groups (P=0.70) (Figure 3A), whereas TIMP-1 did (P<0.0001): it was significantly higher in both pathologies than that in controls and higher in AS compared with AR. The median value of TIMP-1 level was seven times higher in AS than in controls and four times higher in AR than in controls (Figure 3B). The inhibitory capacity of TIMP-1 and TIMP-2 has been measured using reverse gelatin zymography (Figure 3C). The correlation between the measurement of TIMP-1 inhibitory capacity and its protein concentration was significant (r=0.49, P=0.04); the non-significant correlation for TIMP-2 (r=0.35, P=0.21) is probably due to the lack of sensitivity of its detection on reverse gelatin zymography.
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Cell culture
Immunohistological studies showed that interstitial cells in aortic valves and in culture were similarly labelled for smooth muscle--actin and prolyl-4-hydroxylase (Figure 4). Cells stained also positively for vimentin and negatively for desmin and smooth muscle-myosin. Valvular interstitial cell phenotype was identical in valvular tissue and cell culture and was characteristic of myofibroblasts (Figure 4).
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Discussion |
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Histological analysis
At the tissue level, calcification is the most common pathological finding in AS. However, bone formation was observed in only three out of 32 AS valves. Inflammatory cell infiltration, mainly composed of plasmocytes, and the process of angiogenesis were also observed in AS. AR differed from AS mainly by the absence of calcification, the lower content of inflammatory cells and the decreased number of neocapillaries. These results are consistent with previous findings.811
Normal heart valves have a complex layered architecture and highly specialized ECM. The ECM is synthesized by interstitial cells and its composition differs in the three layers. The collagen bundles in the fibrosa provide strength and stiffness to maintain coaptation during diastole. The loose, watery connective tissue of the spongiosa, which contains mainly proteoglycans, confers flexibility and plasticity to the cusp. Elastic fibres present in the ventricularis layer extend during diastole and spontaneously recoil during systole.12,13 Only minor abnormalities of the ECM were observed in our control valves. The extent of disorganization of collagen bundles, the fragmentation and stratification of elastic fibres were significantly increased in AS and AR when compared with controls, but were similar in AS and AR, except the loose connective tissue content which was greater in AR.
Thus, although the pattern of histological abnormalities were different in AR when compared with AS, they shared some common basic ECM abnormalities, raising our interest for the evaluation of the role of MMPs and TIMPs in this process.
Biochemical analysis
Previous studies demonstrated the presence of MMPs and TIMPs in control and pathological valves at the mRNA14,15 and at the protein level.9,1618 However, our study is the first to report a quantitative and comparative analysis of MMPs (MMP-2, MMP-3, MMP-7, and MMP-9) and their tissue inhibitors (TIMP-1 and TIMP-2) in AR and AS human valves.
MMP activities and TIMP concentrations were normalized by the weight of extracted proteins, that is, on a cell protein basis as the ECM components, especially collagens and elastic fibres and calcium minerals are insoluble in the buffers used for extraction. On this cellular basis, the MMP-2 activity was not significantly different between control and pathological valves, that is, the mean MMP-2 quantity secreted per cell was similar in control and pathological valves. Conversely, other studies reported increased quantities of MMP-2 in AS when compared with controls.19,20 These apparent discrepancies can be partly explained by differences in study designs, in particular the expression of MMP-2 activity normalized by the weight of extracted proteins and the use of fresh homografts as controls in the present study.
In the present study, cell culture showed that MMP-2 was constitutively secreted by valvular interstitial cells in culture as it is secreted by other mesenchymal cells such as arterial smooth muscle cells.21 We showed that MMP-2 activity, secreted by valvular cells in culture, was influenced by the concentration of added inflammatory cytokines, as has been shown for arterial smooth muscle cells. Thus, the MMP-2 activity secreted by a cell may be increased or decreased in a pathological valve according to the local concentration of inflammatory cytokine. This could explained the absence of significant difference in the mean MMP-2 activity per microgram of extracted proteins between pathological and control valves (Figure 2A).
Leucocytes (neutrophils, monocytes, and lymphocytes) synthesize small amounts of MMP-2,22 whereas valvular interstitial cells do not secrete MMP-9. This MMP-9 is concentrated in azurophil granules of neutrophils and is synthesized by macrophages.22 The MMP-9 activity was significantly increased in AS compared with AR valves and was higher in AS compared with controls, suggesting a higher degree of inflammation in AS when compared with AR. This is consistent with higher MMP-3 activity and also the significantly increased number of inflammatory cells observed on histological sections of AS. Therefore, in our study, consistent findings of histological and biochemical quantitative analyses suggest a possible link between inflammation and ECM remodelling in aortic valve diseases.
The contribution of the MMP activities to ECM remodelling cannot be investigated without the analysis of their specific inhibitors, the TIMPs. Our results showed that TIMP-1 was significantly increased and TIMP-2 was not modified in AS and AR valves compared with controls. TIMP-1 and TIMP-2 have been detected in arterial endothelial cells and smooth muscle cells.18 In the present study, cell culture showed that TIMP-1 and TIMP-2 were synthesized by valvular interstitial cells. The factors implicated in the regulation of TIMPs have been extensively studied and it has been shown that TIMP-2 is mainly constitutive, whereas TIMP-1 is modulated by a variety of external stimuli such as cytokines, peptides, or hypoxia. The addition of inflammatory cytokines (TNF- and IL-1ß) to valvular interstitial cells in culture did not modify the synthesis and secretion of either TIMP-1 or TIMP-2 (data not shown). One peptide which could modulate TIMP-1 level in pathological valves is angiotensin II because, as angiotensin-converting enzyme, angiotensin II, and the AT-1 receptor have all been detected in sclerotic and stenotic aortic valves23 and angiotensin II induced TIMP-1 production in rat smooth muscle cells.24 Hypoxia is also a potential candidate, alone or in combination with other factors, since angiogenesis, a biological process observed in hypoxic tissue, was observed on histological sections of our pathological valves and hypoxia induces TIMP-1 in pulmonary arteries.25 Whatever the inducer, the increased level of TIMP-1 could limit the ECM remodelling in these end-stage pathological AS and AR valves. A similar increase in TIMP-1 levels has already been demonstrated in hypertensive arteries7 and varicose veins5 in comparison to control vessels, corresponding to a mesenchymal response to various stimuli (hypertension, hypoxia, etc.).
Study limitations
The number of control valves was small. This was the consequence of the deliberate choice to use fresh homografts as controls. The alternative of using autopsy samples as controls would enable age to be matched between pathologic and controls valves, but post-mortem hydrolysis of protein could impair the relevance of the quantitative analysis of MMPs and TIMPs. Another consequence of this choice is that patients with AS were older than both controls and patients with AR.
The analysis of the MMP and TIMP concentrations was performed on protein extracts of valves, resulting in the determination of mean activities and concentrations. Thus, although there was a significant increase in the mean TIMP-1 levels and mean TIMP-1/MMP-9 ratios in AS and AR valves, we cannot exclude that locally the MMP content may exceed the TIMP content, for example, when inflammatory cells are present. Furthermore, as capillaries are detectable in pathological valves, it cannot be excluded that some soluble plasma proteins are present in protein extracts.
This analysis of the ECM remodelling and its molecular effectors was performed in end-stage pathological aortic valves. This limitation is inherent to the analysis of surgically excised human valves. These findings cannot be extrapolated to the pathogenesis of valve disease at an earlier stage.
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Conclusions |
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Acknowledgements |
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References |
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