Ann Thorac Surg 2009;88:1476-1483. doi:10.1016/j.athoracsur.2009.07.025
© 2009 The Society of Thoracic Surgeons
Original Articles: Adult Cardiac
Mechanical Strain and the Aortic Valve: Influence on Fibroblasts, Extracellular Matrix, and Potential Stenosis
Sven Lehmann, MD*,
Thomas Walther, MD, PhD,
Jörg Kempfert, MD,
Ardawan Rastan, MD,
Jens Garbade, MD,
Stefan Dhein, MD, PhD,
Friedrich W. Mohr, MD, PhD
Department for Heart Surgery, Universität Leipzig, Heart Center, Leipzig, Germany
Accepted for publication July 15, 2009.
* Address correspondence to Dr Lehmann, Universität Leipzig, Herzzentrum, Klinik für Herzchirurgie, Strümpellstr 39, Leipzig, 04289, Germany (Email: sven.lehmann{at}med.uni-leipzig.de).
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Abstract
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Background: Mechanical strain may affect aortic valve cusp, leading to an altered extracellular matrix ultrastructure and eventually aortic stenosis. The aim of this study was to evaluate the affect of these potential relationships on human tissue.
Methods: Extracellular matrix protein disposition was analyzed on human aortic valve cusp retrieved from 31 patients during routine aortic valve replacement surgery. Samples were immediately fixed in 2-hydroxyethyl methacrylate. Immunohistology and Western blot analysis were used to quantify decorin, tenascin-C, biglycan, alkaline-phosphatase, osteocalcin, and osteopontin content. Fibroblast function was analyzed on interstitial cells derived from aortic valve cups from patients undergoing aortic valve replacement. Cells were grown to confluency in modified Eagle's medium supplemented with 10% fetal calf serum under sterile conditions. Thereafter, mechanical strain was applied for 72 hours and 60 cycles per minute. Elongation of as much as 10% in comparison with no elongation (control group) was applied. All results were correlated to hemodynamic variables.
Results: Decorin and biglycan were mostly located at the inflow aspects of the cusp, tenascin-C in the central layer, and osteopontin, osteocalcin, and alkaline phosphatase were concentrated near the cell populations surrounding calcified areas. The intensity of this protein expression was significantly related to the pressure gradient. Expression levels were twice to five times higher than normal in patients with a preoperative pressure gradient of more than 100 mm Hg. On fibroblasts subjected to mechanical strain, a similar significant increase in the expression for decorin, biglycan, alkaline-phosphatase, tenascin-C, osteocalcin, and osteopontin was found by immunohistology. Western blot analysis confirmed significantly enhanced expressions of two and eight times the normal levels.
Conclusions: A specific pattern of extracellular matrix protein expression was found in relation to mechanical strain on human aortic valve cusp tissue and in mechanically stimulated human valvular fibroblasts. This new insight into the process of aortic valve degeneration may be important for further therapeutic approaches to ameliorate the progression or even the initiation of potential aortic valve stenosis.
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Introduction
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Nonrheumatic calcified aortic valve stenosis is the most common acquired heart valve lesion in Western societies. It is characterized by degenerative changes with complex calcification of the native cusp and the aortic annulus [1]. The natural history indicates that severe symptomatic aortic stenosis is associated with a life expectancy of less than 5 years [2]. Despite the high prevalence of this condition, little is known regarding the molecular mechanisms leading to aortic valve calcification.
The three distinct layers of the aortic valve cusp are constituted of different types of connective tissue cells, predominantly the extracellular matrix (ECM). The ECM consists of glycoproteins, elastic fibers, fibrillar collagens, and an amorphous substance composed of proteoglycans. The different layers can be separated according to their molecular structure. Functionally, aortic valve cusp can be divided into inflow, central, and outflow layers. Inflow and outflow layers are covered by endothelium. The outflow layer contains collagen fibers running mainly in a circumferential direction. The bulk of the central layer is made up of a watery, loose connective tissue ground substance of varying composition. The inflow layer is relatively thin and has a strong elastin expression. In the outflow layer, spacing around the individual collagen fibrils is quite regular, so they are fixed into some kind of order by the proteoglycans. High-resolution images suggest that these proteoglycans not only fill the spaces, but also contact the outer layer [3]. Human aortic valve cusps consist of 60% collagen, 30% proteoglycan, and 10% elastin. Human heart valves have been shown to contain approximately 60% of their total glycosaminoglycans as hyaluronic acid, 30% as chondroitin-4-sulfate/chondroitin-6-sulfate, and 10% as decorin [4–6].
Proteoglycans are glycoproteins that are heavily glycosylated. They consist of a core protein with one or more covalently attached glycosaminoglycan chains. The chains are long, linear carbohydrate polymers that are negatively charged under physiologic conditions, owing to the occurrence of sulfate and uronic acid groups. Proteoglycans occur in connective tissue of humans. Proteoglycans can be categorized depending upon the nature of their glycosaminoglycan chains. Proteoglycans can also be categorized by size. Examples of large proteoglycans are aggrecan, the major proteoglycan in cartilage, and versican, present in many adult tissues including blood vessels and skin. The small leucine-rich repeat proteoglycans include decorin, biglycan, fibromodulin, and lumican. Proteoglycans are a major component of the human extracellular matrix, the "filler" substance existing between cells in an organism. Here, they form large complexes, both to other proteoglycans, to hyaluronan, and to fibrous matrix proteins (such as collagen). They are involved in binding cations (such as sodium, potassium, and calcium) and water, and also in regulating the movement of molecules through the matrix. Evidence shows they can affect the activity and stability of proteins and signaling molecules within the matrix. Individual functions of proteoglycans can be attributed to either the protein core or the attached glycosaminoglycan chain.
The ECM is an active, dynamic structure that provides tissue integrity together with signaling capabilities to neighboring cells and thus regulatory functions, including cell differentiation. Such regulatory impulses are being transferred by specific receptors or through growth factors. Cultured stretched aortic valve leaflets have a increase in collagen, but a reduced sulfated glycosaminoglycan concentration and no difference in elastin concentration [7]. As such, ECM proteins interact directly with cell surface receptors and initiate signal transduction pathways. Integrins are the major class of ECM receptors. Any modulation from the ECM results in an alteration of their structure and composition through further differentiation of the residing cells. Different ECM components can selectively affect many types of signal transduction pathways, including the suppression of apoptosis or differentiation. Extracellular matrix remodeling is a continuous process with a good balance between component synthesis and breakdown.
In this context, the aim of the study was to analyze ECM components, in particular proteoglycans, in stenosed human aortic valves cusp with respect to flow direction. Furthermore, we tried to elucidate the influence of induced stress on native aortic cusp fibroblasts with regard to production of proteoglycans and ECM.
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Material and Methods
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This study was performed on human samples in the experimental laboratory using two approaches. Tissue ultrastructure was evaluated in stenotic human aortic valve cusp, and the potential influence of induced mechanical strain was evaluated on cultured human aortic valve fibroblasts.
Human tricuspid aortic valves were obtained from patients with aortic valve disease at the time of surgical aortic valve replacement. Informed consent was obtained, and the investigation protocol was approved by the local Ethics Committee.
Tissue Preparation
Valve tissue from 31 patients was immersed in the polymerized medium 2-hydroxyethyl methacrylate (Technovit 8100; Heraeus Kulzer GmbH, Wehrheim, Germany) using controlled temperature embedding. Thin serial sections, 5 µm thick, were cut for staining using a rotational microtome with a hard metal knife. The preoperative patient characteristics and hemodynamic function are given in Table 1.
Cell Culture
Human aortic valves were obtained during valve replacement surgery (n = 20) under sterile conditions. The preoperative patient characteristcs and hemodynamic function of these patients are given in Table 1. Valvular interstitial cells were isolated by outgrowing in Medium 199 with Earle's and GlutaMAX (Gibco, Karlsruhe, Germany) supplemented with 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin (Sigma, Deisenhofen, Germany), 100 µg/mL streptomycin (Sigma) and 6 mg/mL hydroxyethylpiperazine-ethanesulfonic acid (HEPES [Sigma]) at 37°C in humidified air, containing 5% CO2. Fibroblasts were subcultured after adding Trypsin-EDTA solution (Sigma) onto type I collagen-coated flexible-bottom plates (BioFlex Plates; Flexcell International, McKeesport, PA) and incubated at 37°C in a 5% CO2 incubator for 24 hours before applying mechanical strain (Fig 1). The cells were subjected to cyclic strain at 1 Hz (0.5 s of 10% stretch alternating with 0.5 s of relaxation) for different intervals using a computer-controlled vacuum stretch apparatus (FX-4000T Tension Plus System; FlexCell International) with a vacuum pressure that is sufficient to generate 10% mechanical strain. Replicate control samples were maintained under static conditions with no applied cyclic strain.
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Fig 1. Prolyl-4-hydroxylase beta and 3-amino-9-ethylcarbazol method with haemalaun from fibroblast subcultures (black arrow indicates cell nucleus of fibroblast; gray arrow indicates cytoplasm of fibroblast).
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Immunohistochemistry
To determine the expression of protein in ECM cultures, immunohistochemistry was performed by using the peroxydase and 3-amino-9-ethylcarbazol method. The middle part of the flexible-bottom plates was not used. A piece from inside to outside of the flexible-bottom ring was analyzed for every antibody. Cell cultures were fixed with 3.7% paraformaldehyde in phosphate-buffered saline (PBS) overnight. Cell cultures and Technovit 8100 fixed tissue were permeabilized with 0.1% Triton X-100 in PBS for 30 minutes. The endogenous peroxidase activity was quenched by treating the cells for 30 minutes with 3% hydrogen peroxide. To reduce nonspecific binding of the primary antibody, the cells were first blocked for 20 minutes at room temperature with 5% bovine albumine solution (Sigma). The object slide was drained off, and was incubated with the primary antibody against osteocalcin (Santa Cruz Biotechnology, Santa Cruz, CA) with a dilution of 1:100, osteopontin (Santa Cruz Biotechnology) 1:100, alkaline phosphatase (Sigma) 1:4000, decorin (Calbiochem, Darmstadt, Germany) 1:500, biglycan (R&D Systems, Minneapolis, MN) 1:20, and tenascin-C (Santa Cruz Biotechnology) 1:100 overnight at 4°C. The cells were washed thoroughly with Tris-NaCl-Tween 20 (TNT) (three times; each wash for 5 minutes) and blocked for 30 minutes with Tris-NaCl-DuPont Blocking Solution (TNB) and treated with goat anti-rabbit IgG (Dianova, Hamburg, Germany), or rabbit anti-mouse IgG (Sigma) peroxydase conjugate (1:100 in TNB; 1 hour). The cells were then washed with TNT (three times; each wash for 5 minutes), and treated with biotinylated tyramid (Perkin Elmer, Boston, MA [1:50 in amplification diluent, 10 minutes]). The cells were again washed with TNT (three times, each wash for 5 minutes) and were incubated for 30 minutes in streptavidin-horseradish peroxidase (Perkin Elmer [1:100 in TNT]). The cells were washed with TNT one more time for 5 minutes and were treated with 2 drops of 3-amino-9-ethylcarbazol complex for 30 minutes, followed by a thorough washing with PBS (three times, each wash for 3 minutes). Finally, the cells were stained for 5 minutes with distilled water, dried, and observed under light microscopy.
Cells were considered to be positive if brown stain was noted within the cytoplasm above the level of nonspecific signal. The percentage of positive cells was determined at different regions on the membrane.
Western Blot Analysis
For protein extraction, aortic valve tissue samples were homogenized in HEPES buffer with the addition of protease inhibitors (0.5 mg/mL leupeptin, 10 µg/mL aprotinin) and 1 mM phenylmethylsulfonyl fluoride (Boehringer, Mannheim, Germany). A total of 50 µg protein was separated on a 12% sodium dodecyl sulfate polyacrylamide gel and blotted on to nitrocellulose membranes (Roth, Karlsruhe, Germany) with a tank blotting system (BioRad, Munich, Germany). Membranes were blocked with 5% milk powder (Roth) in tris buffered saline with 0.5% Tween 20 for one hour. After washing (three times for 5 minutes in Tween 20 and tris-buffered saline), membranes were incubated with the primary antibodies osteocalcin (Santa Cruz Biotechnology), ostepontin (Santa Cruz Biotechnology), alkaline phosphatase (Sigma), decorin (Calbiochem), biglycan (R&D Systems), tenascin-C (Santa Cruz Biotechnology), and mouse anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH [Hytest, Turku, Finland]) overnight. After a second washing step, membranes were incubated with secondary antibodies conjugated with horseradish peroxidase for one hour, goat anti-rabbit IgG (Dianova), or rabbit anti-mouse IgG (Sigma). Membranes were washed three times in Tween 20 and tris-buffered saline and subsequently developed with Super Signal Reagent (Pierce, Rockford, IL). The specificity of the antibodies has been determined previously.
Immunoblots were exposed to x-ray film (Eastman Kodak, Rochester, NY), developed, and analyzed by Aida 2.0 beta software. The relative amount of osteocalcin, ostepontin, alkaline phosphatase, decorin, biglycan, and tenascin-C receptors in each sample was normalized to the housekeeping protein GAPDH to assure that the same amount of cellular proteins in each sample was determined. The ratio of primary antibody receptors/GAPDH from each assay was used to calculate possible differences in primary antibody receptor synthesis between the samples.
In a first step of statistical analysis, analysis of variance (ANOVA) was performed. If ANOVA indicated significant differences, the data were additionally analyzed with a t test, Mann-Whitney U test, and Pearson correlation test. A p value less than 0.05 was considered to indicate statistical significance. For statistical analysis, SPSS 13.0 (SPSS, Chicago, IL) was used, and for the figures, SigmaPlot software was used. Patient characteristics variables are expressed as mean ± SD and the protein expression are given as mean ± SEM of n experiments.
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Results
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Immunohistology revealed an upregulation of osteocalcin, alkaline phosphatase, tenascin-C, and biglycan in relation to the severity of aortic valve stenosis. All variables revealed a significant correlation with the preoperative pressure gradient. This is shown on Figure 2
for osteocalcin (Fig 2A), ostepontin (Fig 2B), alkaline phosphatase (Fig 2C), tenascin-C (Fig 2D), decorin (Fig 2E, Fig 3A and B), and biglycan (Fig 2F) in relation to the transvalvular pressure gradient.
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Fig 2. (A) Osteocalcin expression in relation to the preoperative maximum transvalvular pressure gradient. (B) Osteopontin expression in relation to the preoperative maximum transvalvular pressure gradient. (C) Alkaline phosphatase expression in relation to the preoperative maximum transvalvular pressure gradient. (D) Tenascin-C expression in relation to the preoperative maximum transvalvular pressure gradient. (E) Decorin expression in relation to the preoperative maximum transvalvular pressure gradient. (F) Biglycan expression in relation to the preoperative maximum transvalvular pressure gradient.
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Fig 3. (A) Immunohistochemistry of decorin expression in human aortic valve with a preoperative maximum transvalvular pressure gradient from 60 mm Hg. (1 = inflow layer; 2 = central layer; 3 = outflow layer; black arrow = decorin.) (B) Immunohistochemistry of decorin expression in human aortic valve with a preoperative maximum transvalvular pressure gradient from 120 mm Hg. (1 = inflow layer; 2 = central layer; 3 = outflow layer; black arrow = decorin.)
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The highest upregulation in relation to the transvalvular pressure gradient was found for decorin (Fig 3A and B) and biglycan, and the lowest, but still significant, increase for osteocalcin and alkaline phoshatase.
The expression of decorin and biglycan was more intense in the inflow domain of the cusp with a lower concentration in the other layers (Fig 3B). There was a limited expression in the area of coaptation. The highest concentration from decorin and biglycan was found in the middle of the leaflets. Tensacin-C was concentrated in the central layer and showed a more diffuse distribution in the outer layers. In the coaptation area of the cusp, there was a limited expression of tenascin-C. The highest concentration of tenascin-C was found in the middle of the leaflets. Osteopontin, osteocalcin, and alkaline phosphatase were concentrated near the cell populations surrounding calcified areas.
Seventy-two hours of stretch on cultured aortic cusp fibroblasts resulted in significant upregulation of the same proteins that were identified in heavily calcified tissue. As such, a significant upregulation of osteocalcin (Fig 4A and B), ostepontin, alkaline phosphatase, tenascin-C, decorin, and biglycan were found (Fig 5). The higest upregulation was found for decorin, biglycan, osteocalcin (Fig 4A and B), and alkaline phosphatase. The lowest upregulation, still significant, was found for ostepontin and tenascin-C.
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Fig 4. (A) Immunohistochemistry of osteocalcinexpression after 72 hours without stress (double black arrow indicates fibroblast; black arrow indicates osteocalcin). (B) Immunohistochemistry of osteocalcinexpression after 72 hours with stress (double black arrow indicates fibroblast; black arrow indicates osteocalcin).
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Quantitative Western blot analysis confirmed these results and also showed significant upregulation of osteocalcin, ostepontin, alkaline phosphatase, tenascin-C, decorin, and biglycan (Fig 6). The highest upregulation in response to stretch was found for decorin and osteocalcin. The lowest upregulation was found for alkaline phosphatase and tenascin-C (Fig 7).
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Fig 6. Quantitative Western blot analysis against biglycan and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (Line 1 = after 72 hours of stress; line 2 = without stress.)
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Fig 7. Protein expression without stress (black bars) and after 72 hours of stress (gray bars) in relation to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
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Comment
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The causes for initiation and progression of aortic valve stenosis still are not fully understood. Therefore, we performed systematic experimental evaluations on (1) the molecular composition of human stenotic aortic valve cusp, and (2) the potential influence of mechanical strain on human aortic valve fibroblasts. This, together with hemodynamic parameters of the patients, enabled us to correlate molecular changes to clinical findings.
The most important finding of the present analysis was that similar upregulation of extracellular matrix proteins, in particular proteoglycans, was found on human cusp in relation to the pressure gradient as well as on cultured fibroblasts in relation to the mechanical strain applied. These findings are in accordance with the general assumption of these proteins are involved in the process of aortic valve calcification and stenosis. Proteoglycans play an important role throughout the body regarding tissue mechanics, tissue hydration and the regulation of extracellular calcium homeostasis. Mechanically, their large molecular weight, as well as anionic and hydrophilic nature, allow them to function as excellent lubricants and shock absorbers. Their ability to perform these functions has been extensively investigated with particular attention to the synovial joints such as the knee [8, 9]. In addition, their proposed function in the regulation of new bone formation by virtue of their ability to create positively charged calcium ions has also been widely studied [10–14]. Despite their relative abundance within the matrix of heart valves, however, the potential for similar mechanical and biochemical functions within these tissues has only recently been proposed, and there is very little evidence thus far [15–18]. The present analyses add some evidence to underline the involvement of proteoglycans in heart valve calcification.
In normal porcine cultured stretched aortic valve leaflets, Balachandran and colleagues [7] found an increase in collagen, but a reduced sulfated glycosaminoglycan concentration and no difference in elastin concentration. On left-sided (mitral and aortic) ovine heart valves, Merrymann and coworkers [6] found significantly stiffer tissue in comparison with right-sided valves. In comparison with those findings, we studied human aortic valve samples.
Recent research has revealed striking similarities between the cellular and molecular mechanisms controlling heart valve cell differentiation and those of developing cartilage, tendon, and bone [12, 14]. It is likely that these findings do not represent the full regulatory conservation of these tissue types, and that additional pathways originally characterized in developing cartilage, tendon, and bone will be found to be important for heart valve lineage differentiation and remodelling. For example, there is initial evidence that Notch and canonical Runx2 signaling pathways are active during valve remodeling, but the specific cells and processes affected by these pathways are not well characterized [19, 20]. Insights into the functions of these pathways in the valves could be provided by extensive mechanistic studies of cartilage, tendon, and bone development [19, 20]. The conservation in genetic mechanisms between these systems has certainly advanced our understanding of heart valve remodeling thus far and will likely provide exciting fields for further research in future.
There is an increasing understanding of the specific connective tissue cell types constituting the different structures of heart valves. In addition, there is emerging evidence that dysregulation of these cell types is associated with valve disease. Transcriptional regulators, such as Sox9, scleraxis, and NFATc1, are expressed during valve lineage differentiation and patterning, but further studies are necessary to determine the precise functions of these genes during valvulogenesis in vivo [3, 19, 21]. It is tempting to speculate that these critical regulators of valve development also play a role in aberrant ECM expression and organization associated with valve disease. However, induction of these factors during valve pathogenesis has not yet been demonstrated. In addition, appropriate viable animal models for molecular and cellular studies of myxomatous or calcified valves are not widely available. Human genetic studies are beginning to provide evidence that developmental pathways important in valvulogenesis also contribute to adult valve disease [3, 12, 14, 19, 21].
In the present study, stress led to an increased induction of ECM proteoglycan expression. It is of further note that all proteoglycans had an acid end. The upregulation of negative ends leads to an increase in binding sites for potential adsorption of calcium. This may be an alternative pathway to explain calcification of the aortic valve cusp, at least in the early stages. Interestingly, a parallel finding in atherosclerosis research has been published demonstrating the involvement of proteoglycans as Ca++-binding components in the earliest plaque formation of the so-called nanoplaques [22, 23].
Nevertheless, more research is required to determine whether valve degeneration that can be characterized by abnormal or mineralized ECM is in fact caused by reactivation of developmental gene programs and if these molecular mechanisms can be exploited for future therapeutic approaches.
Besides such potential mechanisms, blood flow may have an additional modulator effect on the expression of proteoglycans and extracellular matrix proteins. In fact, we were able to prove that the characteristic pathologic findings were discrete, with focal lesions on the aortic side of the cusp, which can extend deeply into the aortic annulus. Typically, there is displacement of the subendothelial elastic layer by protein and lipid infiltration in conjunction with cellular infiltration with macrophages and T lymphocytes. Gradual increase in size and calcification of subendothelial lesions leads to increased valve stiffness and increasing valvular obstruction [14, 24–26]. Both diabetes mellitus and hypercholesterolemia are risk factors for the development of degenerative calcific aortic stenosis. Aortic sclerosis as well as calcific aortic stenosis is associated with traditional risk factors for development of atherosclerosis: hypertension, cigarette smoking, and hypercholesterolemia [27]. Acquired aortic stenosis is associated with some other less frequent conditions, including end-stage renal failure, Paget disease of the bone, and rheumatic fever [28–30]. Ochronosis is a rare cause of isolated aortic stenosis, which can also cause a rare greenish discoloration of the aortic valve [31].
A limitation of the cell culture study is that the silicone matrix in the cell culture can have a different effect on the monoclonal cell culture. In the human aortic valve, we have different cell types and zonal formations. To circumvent this problem, however, we decided to use collagen-I-coated silicon matrix.
In summary, the present results may represent an early process by which stretch or pressure gradient can induce an upregulation of proteoglycans, which in turn can be the initiating process for ongoing calcification of the aortic valve and the development of aortic stenosis.
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Abstract
Introduction
