Ann Thorac Surg 2003;76:681-688
© 2003 The Society of Thoracic Surgeons
Original article: cardiovascular
Evidence for an altered balance between matrix metalloproteinase-9 and its inhibitors in calcific aortic stenosis
Jari Satta, MD, PhDa*,
Jani Oiva, MSa,
Tuula Salo, MD, PhDb,
Heidi Eriksen, MDc,
Pasi Ohtonen, MSa,
Fausto Biancari, MD, PhDa,
Tatu S. Juvonen, MD, PhDa,
Ylermi Soini, MD, PhDd
a Departments of Cardiothoracic Surgery, Oulu, Finland
b Diagnostics and Oral Medicine, Institute of Dentistry, Oulu, Finland
c Clinical Chemistry,, Oulu, Finland
d Pathology, University of Oulu and Oulu University Hospital, Oulu, Finland
Accepted for publication March 10, 2003.
* Address reprint requests to Dr Satta, Department of Cardiothoracic Surgery, PO Box 5000, 90014 University of Oulu, Oulu, Finland
e-mail: jari.satta{at}oulu.fi
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Abstract
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BACKGROUND: Recently, aortic valve stenosis has been demonstrated to exhibit increased expression of certain matrix metalloproteinases (MMPs), and this has relevantly raised the question about possible interdependency between these and their tissue inhibitors. We sought to assess the expression of elastolytic MMPs and their inhibitors (TIMPs) in nonrheumatic aortic stenosis.
METHODS: The study comprised 30 stenotic and six noncalcified human aortic valves. To measure the expression levels and the amount and molecular forms of gelatinases (MMP-2, MMP-9) and TIMPs (1, 2), in situ hybridization, gelatin zymography, and reverse zymography were carried out. Antielastin staining by a monoclonal BA-4 antibody was performed to investigate the changes of one of the main substrates of these MMPs, and to substantiate the nature of the putative MMP- synthesizing cell. The cases were also immunostained with an antibody to 
-smooth muscle actin. Inflammatory cell characterization was managed by monoclonal mouse antibodies (UCHL-1, L26, and PGM-1).
RESULTS: Compared with the controls, the calcific valves showed increased mRNA expression and activation of MMP-9, and this was associated with typical characteristics of valve disease. MMP-2 mRNA production was rare, but proMMP-2 protein was detected in all valves. In agreement with the interdependency between MMP-9 and its inhibitors, a suggestive imbalance came out in diseased valves.
CONCLUSIONS: The disproportion between MMP-9 and its tissue inhibitors may favor a persistent MMP activation state within the calcific valve and likely contribute to the valvular remodeling process in the setting of developing aortic stenosis.
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Introduction
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The importance of matrix metalloproteinases (MMP) in the pathogenesis of cardiovascular disorders is being increasingly recognized, particularly with respect to atherosclerotic lesions, aortic and cerebral aneurysms, and heart failure [1, 2]. Recently, certain MMPs have been also closely connected to the pathogenesis of aortic valve stenosis (AS) [3–5].
MMPs constitute a family of endopeptidases that have in common the presence of zinc at their active site, dependency on Ca2+ for their activity, and an ability to react with specific tissue inhibitors (TIMPs) to form enzymatically inactive complexes [6]. A balance between MMP and TIMP activities is a prerequisite for normal function of an array of physiologic processes, and disruption of this balance may result in diseases associated with uncontrolled turnover of extracellular matrix (ECM). MMPs show a wide range of specificity for different substrates, including native and partially degraded fibrillar collagens, basement membrane collagens, proteoglycans, elastin, and fibronectin. In terms of the cardiovasculature, the ability of certain MMPs, such as MMP-2, MMP-3, and MMP-9, to hydrolyze elastin is of particular importance.
The purpose of the present study was to investigate the mRNA synthesis and gelatinolytic activity of elastolytic MMPs (MMP-2 and MMP-9) in AS and especially to correlate their expression with their tissue inhibitors (TIMP-1 and TIMP-2) during the progression of the disease.
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Material and methods
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Patients
Thirty tricuspid, nonrheumatic stenotic aortic valves (13 men; mean age, 71 years; range, 58 to 82 years) were collected from patients undergoing aortic valve replacement because of AS. Most of the patients underwent the operation because of isolated valve stenosis, and some of them underwent a combination procedure, usually involving coronary bypass grafting, in cases of milder valve pathology, and this enabled us to obtain samples from a relatively comprehensive range of disease conditions. The classification of disease severity into mild (n = 8), moderate (n = 10), and severe (n = 12) was based on the ACC/AHA guidelines supported by histopathological findings [7, 8]. For controls, six macroscopically normal, noncalcified, smooth, and pliable tricuspid valves were collected from non-Marfan individuals operated on mainly because of aortic root pathology. Samples were taken vertically through the leaflet near the central part, and immediately after removal, the valves were fixed in formaldehyde and then embedded in paraffin. For zymography and reverse zymography, 12 stenotic and six control valves were rinsed with physiologic saline, and within half an hour from excision, transferred into liquid nitrogen and stored at –70 o C until used.
Characterization of the probes
A 635-bp Sca I–Sac I fragment of the K-191 cDNA clone coding for human MMP2 [9] was ligated into the M13 polylinker site of pSP64 and pSP65 vectors (Promega, Madison, WI). The fragment spanned the nucleotides 578 to 1213 of the human MMP2 cDNA sequence. A 574-bp Eco RI–Hind III fragment of the human MMP9 cDNA clone K-174.1 [10] was subcloned into the pGEM 4Z vector (Promega). The restriction sites were 1751 and 2325. A 626-bp Bam HI–Hind III fragment of the TIMP1 coding sequence [11] spanning the nucleotides 62 to 688 of the TIMP1 cDNA was cut from the pUC 19 vector and ligated in pGEM 4Z (Promega). A 388-bp Eco RI–Kpn I fragment of the TIMP2 cDNA clone [12] extending from nucleotides 710 to 1098 from the SS 38 vector was ligated in pGEM 4Z (Promega).
In situ hybridization
Before hybridization, the sections were deparaffinized in xylene and dehydrated in an ethanol series. The sections were then treated with 0.2 mol/L HCL for 20 minutes at room temperature (RT) and washed in DEPC-H2O for 5 minutes, after which they were treated with proteinase K (1 mg/mL) for 30 minutes at 37oC. They were then incubated in 0.2% glycine in phosphate-buffered saline (PBS) and washed twice in 1x PBS for 30 seconds. The sections were then postfixed with 4% paraformaldehyde in PBS for 20 minutes, washed in 1x PBS, and acetylated in 0.25% to 0.50% acetic anhydride in 0.1 mol/L triethanolamine for 10 minutes, rinsed in 1x PBS, dehydrated, and air-dried for 1 to 2 hours at RT. After this, the sections were treated with prehybridization mixture for 2 hours (10 mmol/L DDT, 10 mmol/L Tris-HCl, 10 mmol/L NaPO4, 5 mmol/L EDTA, 0.3 mol/L NaCl, 1 mg/mL Yeast tRNA, deionized formamide 50%, and dextran sulfate 10% [w/v]; 0.02% [w/v] Ficoll, 0.02% [w/v] polyvinylpyrrolidone, and 0.02 mg/mL bovine serum albumin). They were then washed in 1x PBS and dehydrated. In the hybridization step, the probes were first denatured by boiling for 1 minute and then placed on ice. A total of 3 x 106 cpm of the 35S-labeled antisense or sense probe in 40 µL prehybridization buffer was applied to each section, and the hybridization was carried out at 50oC overnight. The posthybridization washes were performed as follows: the specimens were washed twice at 50oC for 1 hour in prehybridization mixture, except for dextran sulfate and tRNA, rinsed for 15 minutes in 0.5 mol/L NaCl in 10 mmol/L Tris-HCl, 1 mmol/L EDTA (TE) at 37oC, and then incubated for 30 minutes in 0.5 mol/L NaCl in TE containing 40 µl/mL RNase A (Sigma, St. Louis, MO) at 37oC, washed for 15 minutes in 0.5 mol/L NaCl in TE at 37oC, twice for 15 minutes in 2x standardized saline citrate (SSC), and twice for 15 minutes in 1xSSC, both times at 50oC. The sections were dehydrated in graded a series of ethanol containing 300 mmol/L ammonium acetate and air dried at RT for 1 hour. The slides were then subjected to autoradiography by dipping them into NTB-2 film emulsion (Kodak) and placed in light-tight boxes for 10 to 14 days. The slides were developed in D-19 developer (Kodak), fixed in Agefix (Kodak), and counterstained in hematoxylin-eosin.
The results were assessed on a semiquantitative scale as follows: – = no signals present; + = only weak mRNA signals present; ++ = moderate mRNA signals present; +++ = strong mRNA signals present.
Immunohistochemistry of elastin and cellular composition
For immunophenotypic analysis of inflammatory cells, monoclonal mouse antibodies UCHL-1, L26, and PGM-1 (at a dilution of 1:50) (Dako, Glostrup, Denmark) were used. The extent of inflammation was semiquantified as none, weak (+), moderate (++), or severe (+++) according to the number of inflammatory cells. The inflammatory infiltrate was considered weak if only a few scattered lymphocytes could be found in valvular tissues, mainly detected in high-power views. It was considered moderate if lymphocytes were easily found in low-power views. The lymphatic infiltrate was considered strong if the lymphocytes formed well-recognized groups of inflammatory cells in quantities exceeding 50 cells/HPF.
To identify smooth muscle cell differentiation in fibroblast type cells, commercially available antibodies against -smooth muscle actin (Clone 1A4, Sigma Biosciences, St. Louis, MO) were used at a dilution of 1:1000. To detect elastin in tissue sections, a monoclonal antielastin BA-4 antibody was used at a dilution of 1:100. This antibody has been raised against an elastin-derived chemotactic peptide and characterized previously using solid-phase enzyme-linked immunosorbent assay, Western blot, immunoprecipitation, and immunoaffinity chromatography [13]. The intensity of elastin immunoreactivity was assessed on a semiquantitative scale as follows: +++ = normal elastin content; ++ = moderate elastin content; + = mild elastin content.
For immunostainings, the avidin-biotin peroxidase method was used. As a negative control, PBS was used instead of the primary antibody.
Characterization of the extent of calcification and fibrosis
The extent of calcification and fibrosis was classified as follows: mild (+) if less than 25%; moderate (++) if 25% to 50%; and severe (+++) if more than 50% of the valvular area contained fibrotic or calcific tissues.
Zymography
Gelatinases (MMP-2 and MMP-9) were assayed from dry-weighed homogenized tissue extracts suspended into PBS-Tween 20 (0.4%), pH 7.2, to a final concentration of 20 mg/mL, homogenized by sonication for four times for 15 seconds in an ice bath, incubated in ice for 30 minutes, and centrifuged (8,000 g for 30 minutes). The supernatants corresponding to the soluble tissue extracts were collected for further analyses. The total protein content of the soluble tissue extracts was measured by a Bio-Rad DC Protein Assay Kit (Bio-Rad, Hercules, CA) on a microplate following the manufacturer’s instructions. Fifteen microliters (0.3 mg/mL of total protein) of the soluble tissue extract samples pooled from 5 µL of three representative stenotic (pool S) and three control valves (pool C) were mixed with sample buffer [14] and electrophoresed as described before [15] using gelatin zymography, where 10% SDS-polyacrylamide gel was impregnated with 1 mg/mL gelatin fluorecent-labeled with 2-methoxy-2, 4- diphenyl-3 [2H] furanone (Fluka, Ronkonkoma, NY). This method allows for visual monitoring of gelatin degradation under long-wave ultraviolet light during incubation. After electrophoresis, the gels were washed in 2.5% Triton X-100 twice for 30 minutes to remove SDS. The gels were then incubated in 50 mmol/L Tris, pH 7.5, 5 mmol/L CaCl2, 1 µmol/L ZnCl2 at 37°C overnight and photographed. Prestained molecular weight standards and purified MMP-2 and -9 aliquots from keratinocyte culture media were used as controls.
Reverse zymography
TIMPs were assayed using 12 µL of the same soluble tissue extract pools (S and C) as for the zymography above (three stenotic and tree control samples, each pool containing 15 µg total protein) and 10 ng of recombinant TIMP-1 (a generous gift from Dr Timo Sorsa, University of Helsinki, Finland), and prestained molecular weight standards were mixed with sample buffer [14] and electrophoresed using fluorescent-labeled gelatin reverse zymography containing 50 ng recombinant MMP-2 (Sigma, Steinheim, Germany) and 50 ng recombinant MMP-9 (Oncogene, Darmstadt, Germany) according to the method describe by Oliver and associates [16]. After electrophoresis, the gels were washed and incubated at 37°C overnight and photographed over an ultraviolet light box. Areas of gelatinolytic inhibition were visualized as black bands of the reverse zymogram on a clear background.
Statistical analysis
Nonparametric Kendall’s rank correlation coefficient (
) was calculated to measure validity between the grading of aortic valve stenosis, the enzyme signals, and the characteristics typical of valve pathology. Analyses were performed using a standard statistical program (SPSS 10.0, SSPS Inc, Chicago, IL).
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Results
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In terms of MMP-9, TIMP-1, and TIMP2 mRNA quantity, in situ hybridization revealed important differences between the controls and stenotic valves, and these results are shown in Figure 1.
Regarding MMP-2 mRNA expression, only weak signals could be detected in four of 12 of the cases with severe forms of the disease. As to MMP-9 transcripts, the strongest signals were detected in the fusiform myofibroblast-like cells adjacent to the rims of the calcific lesions (Fig 2A),
whereas less intense signals were seen in cells in the fibrotic areas and in the endothelium. In the control valves, ECM presented a balanced situation implied by the weak or absent mRNAs of MMP-9 and its inhibitors (Fig 2B, 2C). In AS, the mRNA expression of MMP-9 correlated positively with the degree of valve stenosis ( = 0.73, p = 0.001) as well as with the characteristics typical of valve pathology, that is, fibrosis and calcification ( = 0.48, p = 0.001 and = 0.75 p = 0.001, respectively) (Fig 3).
When osseous metaplasia was present, MMP-9 positivity was seen in osteocytes. In contrast to this, TIMP-1 and TIMP-2 hybridization signals had a negative correlation with the degree of disease severity ( = -0.66, p = 0.001 and = - 0.59 p = 0.001, respectively), and a comparison of their transcripts’ amount with that of MMP-9’s during the course of the disease showed significant contrast ( = -0.43, p = 0.008 and = -0.54, p = 0.001). When expressed, signals of TIMP-1 and TIMP-2 produced by fusiform cells were evident adjacent to the calcific lesions (Fig 2D).
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Fig 1. During the progression of aortic stenosis (mild, moderate, and severe), in situ hybridization revealed imbalance in MMP9 (black bars), TIMP1 (grey bars), and TIMP2 (striped bars) production. The x-axis shows semiquantitatively scaled transcript intensity (0 = no signals, + = only weak signals, ++ = moderate signals, and +++ = strong signals), and the y-axis shows the proportional percentage of valves in each classified expression level.
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Fig 2. Hematoxylin & eosin–stained calcified aortic valve samples shown by in situ hybridization strong signals to MMP-9 transcripts in the fusiform myofibroblast-like cells (antibody to -smooth muscle actin) (arrows) (A), contrary to randomly seen signals for TIMP-1 (and much less for TIMP-2) (arrow) (D). Noncalcified valves expressed "steady-state" milieu by no signals for MMP-9 or its tissue inhibitors (B, C). BA-4 immunostaining by the avidin-biotin method expressed elastin in noncalcified valves as an intense and thin lamellar (arrow) zone beneath the surface endothelial cells (E), whereas diseased valves expressed scattered and nonuniform BA-4 immunopositivity (arrow) beneath the basement membrane and in the stroma adjacent to a calcific deposit (F). Magnification: A, D = x160; B, C, E, F = x110.
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Fig 3. During mild to severe progression of aortic stenosis, the production of MMP9 (black bars) correlated positively with valve calcification (grey bars). In regard to BA4 (striped bars) immunopositivity (elastin content), the correlation was negative. On the x-axis are presented semiquantitatively determined MMP9 expression level (0 = no, + = weak, ++ = moderate, +++ = strong signals), extent of calcification (0 no calcification, + if < 25%, ++ if 25% to 50%, +++ if > 50% of the valvular area contained calcific tissues), and intensity of elastin immunoreactivity (+++ = normal elastin content, ++ = moderate elastin content, + = mild elastin content). The y-axis shows the proportional percentage of valves in each determined group.
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In zymography, the stenotic valve samples expressed 92- and 72-kDa bands corresponding to the latent proforms of MMP-9 and MMP-2, respectively. In addition, the stenotic valves produced a 82-kDa active form of MMP-9. The control valve samples contained proMMP-2 and a small amount of proMMP-9, but not active MMP-9 (Fig 4A).
In reverse zymography, the control valves revealed both TIMP-1 and TIMP-2 protein bands, whereas only TIMP-1 was visible in the stenotic sample pool (Fig 4B).
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Fig 4. MMP-9 and MMP-2 in tissue extracts of stenotic (S) and control (C) aortic valve pools analyzed by zymography. The stenotic valves contained both pro- and active MMP-9 forms (92 and 82 kDa, respectively), and pro-MMP-2 (72 kDa). Controls had only a faint pro-MMP-9 and a clear pro-MMP-2 band. Pro-MMP-9, active MMP-9, and pro-MMP-2 are marked by arrows on the right side of the figure (A). By reverse zymography, calcified valves contained only TIMP-1, whereas control valves produced both TIMP-1 and TIMP-2 proteins. TIMP-1 (28.5 kDa) and TIMP-2 (21 kDa) are marked by arrows on the right side of the figure (B). Molecular mass (in kDa) standards are marked on the left.
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In nondiseased valves, BA-4 staining emphasized the intense continuous laminated elastic layer beneath the endothelium and the thinner layer in the ventricular aspect of the valve (Fig 2E). Along the progression to severe alterations, the intensity of immunostaining became weaker and its distribution was scattered and nonuniform ( = - 0.68, p = 0.001) (Fig 2F). Regarding the inverse changes between MMP-9 mRNA and its substrate during the disease development, the association was significant ( = - 0.52, p = 0.001) (Fig 3).
Mononuclear inflammation was a pronounced feature in all of the stenotic valves analyzed. The majority of these cells expressed a T phenotype, and a slight degree of CD68-positive macrophages was also present. Despite the distinct inflammatory cell infiltration in the moderate and severe forms of valve disease, the association with MMP-9 transcript expression did not reach statistical significance (p = 0.1).
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Comment
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In regard to elastolytic MMPs in advanced AS, the present study suggests a significant shift towards MMP-9 mRNA and protein expression, its actual activation, and possible disruption of the balance between it and its inhibitors during the progression of the disease. Its detailed function in AS still remains obscure, but in regard to its function in other cardiovascular pathologies, it may be implicated in several aspects of the pathogenesis of AS, including ECM degradation, cell migration, calcification, and angiogenesis. The main synthetic activity of MMP-9 was presented by fibroblasts/myofibroblasts, endothelial cells, and occasionally osteocytes, suggesting that precisely these cells are responsible for tissue damage/remodeling in AS. The rare mRNA signals of MMP-2 indicated its low actual synthesis rate at the time of retrieval, but the fact that proMMP-2 was evident in stenotic valves may be explained by the observations by Jian and associates [4]. They also detected only pro-MMP-2 in calcific cusps, but when aortic valve interstitial cells were cultivated on collagen supplemented with tenascin-C (TN-C), both MMP-2 mRNA expression and MMP-2 gelatinolytic activity were upregulated. MMP-2 is known to be activated by cell membrane–associated MMP, MT-1-MMP [17], but calcific lesions as hypoxic and hypocellular regions lack the capacity to form MT-1-MMP, contributing to a low activation rate of MMP-2. Thus, the MMP-2 and presumably TIMP-1 proteins detected in clinical aortic valve retrievals could have been secreted and laid down on the matrix during the years preceding surgery, and they may have had an active role in matrix turnover in the early phases of the disease. In noncalcified valves, the expressed levels of gelatinases and their inhibitors were very low or even absent, but the tissue extract pools enclosed the corresponding proteins. This may suggest that although in the "steady-state milieu" gelatinase and their inhibitor mRNAs are not actively transcribed, the proteins may, with time, enter from the serum or become continuously accumulated during the normal tissue turnover process. Most likely, the gelatinases in the control valves are not catalytically active because neither MMP-2 or MMP-9 was visualized in their catalytically active, lower molecular weight forms. In stenotic valves, only a faint band of TIMP-1 and no TIMP-2 was present in reverse zymography, but MMP-9 was detected also in an active 82-kDa form, supporting the assumption of the imbalance between MMP-9 and its inhibitors, as detected also by the corresponding mRNA signals during the progression of AS.
Usually, upregulation of MMPs is assumed to be associated with similar upregulation of TIMPs, but in AS, the regulation seems to be reciprocal. Thus, certain factor(s) potentially exist in AS that may cause overexpression of MMPs to a greater degree than TIMPs, contributing to the pathology towards grossly distorted valve cusps with calcification and degeneration of the most ordinary structural components. Hypothetically, in end-staged AS, hypoxic milieu produced by heavy degenerative alterations may be one such a factor. In agreement with this, Romanic and associates found in their very recent experiments that after chronic permanent myocardial ischemia, MMP-9, -3, and -2 were significantly upregulated, whereas the expression of TIMP-1 was repressed [18]. Similarly, when examining whether changes in oxygen levels affect TIMP and MMP expression in cultured trophoblast and breast cancer cells, Canning and associates found, under reduced oxygen conditions, a distinct shift to the expression of MMP-9 compared with TIMP-1 expression, and this was associated with increased cellular invasiveness [19].
Pathologic calcification is a major terminal process in a variety of cardiovascular diseases, also including AS. In particular, elastin present in cardiovascular structures is known to be prone to mineralization, and in this context, both present suggestive MMP-9/TIMP imbalance as well as our previous finding concerning abnormal tenascin-C (TN-C) expression in AS [20] may have important contributions. Elastin-derived peptides generated by gelatinase degradation of elastic fibers are chemotactic in nature and stimulate cell proliferation, calcium ion influx modification, as well as MMP secretion. In particular, calcium binding to these peptides initiates elastin calcification. Vyavahare and associates used their rat subdermal implant model to nicely point out the significant contribution of gelatinases and TN-C in elastin-oriented calcification [21]. To summarize their findings, both MMPs and TN-C were expressed within elastin implants at an early stage of calcification, and thereafter, in vivo administration of a MMP inhibitor caused a significant reduction in elastin implant calcification with an associated reduction in TN-C, indicating that MMPs are important mediators of both TN-C production and elastin-oriented calcification.
As in calcified atherosclerotic lesions, angiogenesis has also been recently postulated to be an important pathologic implicator in calcific aortic valves [20]. Remodeling of the ECM is a crucial event during angiogenesis, and the importance of MMPs (MMP-1, MMP-2, MMP-9, MT1-MMP) secreted by endothelial cells in this context is proved by the evidence that endogenous and synthetic inhibitors of these enzymes block the process and by the impaired angiogenic response in mice with MMP deficiency [6]. Proteases enable endothelial cells to migrate through the basement membrane into the interstitial stroma and participate in the final organization of the cells into tubular structures. Apart from this role in the breakdown of connective tissue barriers, proteases and their inhibitors are essential for endothelial cell attachment, proliferation, survival, and migration, acting either directly on cells or releasing matrix-associated angiogenic factors and active fragments of matrix components. However, during the maturation of newly formed vessels, the balance between MMPs and their inhibitors must be restored to favor basement membrane assembly and endothelial cell differentiation and quiescence [6]. Hence, extrapolating this to the progression of valve disease, we can postulate that the angiogenic response is probably fixed to the late mild to moderate phases of the disease when the expression of MMP and its inhibitors is fairly balanced (see Fig 1).
Conclusions
The present findings suggest that MMP-9 contributes to matrix remodeling in the late phases of AS, and that this may be attenuated by a disruption of the balance between MMP-9 and its inhibitors. The elucidation of upstream signaling mechanisms that contribute to the selective induction of MMP-9 and presumably other MMPs within the aortic valve, as well as strategies to normalize the balance between MMPs and TIMPs, may yield some therapeutic alternatives by which to control valvular remodeling and thereby slow the progression of the AS process.
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Abstract
Introduction
