PRE-CLINICAL RESEARCH
Endothelium-Dependent Regulation of the Mechanical Properties of Aortic Valve Cusps
Ismail El-Hamamsy, MD*,
Kartik Balachandran, MS ,
Magdi H. Yacoub, FRS*,*,
Louis M. Stevens, MD, SM ,
Padmini Sarathchandra, PhD*,
Patricia M. Taylor, PhD*,
Ajit P. Yoganathan, PhD and
Adrian H. Chester, PhD*
* Tissue Engineering Department, Harefield Heart Science Centre, National Heart and Lung Institute, Imperial College London, Harefield, United Kingdom
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia
School of Public Health, Harvard University, Boston, Massachusetts
Manuscript received September 3, 2008;
revised manuscript received November 5, 2008,
accepted November 6, 2008.
* Reprint requests and correspondence: Sir Magdi H. Yacoub, Harefield Heart Science Centre, Hill End Road, Harefield, Middlesex UB9 6JH, United Kingdom (Email: m.yacoub{at}imperial.ac.uk).
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Abstract
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Objectives: The aim of this study was to evaluate the role of valve endothelium in regulating the mechanical properties of aortic valve cusps.
Background: Mechanical properties of valve cusps are key to their function and durability; however, little is known about the regulation of valve biomechanics.
Methods: Mechanical properties of porcine aortic valve leaflets were evaluated in response to serotonin (5-hydroxytryptamine [5-HT]), with and without N-nitro-L-arginine-methyl-ester (L-NAME) or endothelial denudation, and endothelin (ET)-1, with and without cytochalasin-B.
Results: Under physiological loading conditions, 5-HT induced a decrease in the areal stiffness of the cusp (–25.0 ± 4.0%; p < 0.01 vs. control), which was reversed by L-NAME or endothelial denudation (+17.5 ± 5.3%, p = 0.07, and +14.7 ± 1.8%, p < 0.05 vs. control, respectively). ET-1 caused an increase in stiffness (+34.4 ± 13.8%; p < 0.05 vs. control), but not in the presence of cytochalasin-B (p = 0.29 vs. control). Changes in cusp stiffness were accompanied by aortic cusp relaxations to 5-HT (–0.29% ± 0.1% change in load per 10-fold increase in 5-HT concentration; p = 0.03), which were reversed by endothelial denudation (+0.29 ± 0.06% change in load per 10-fold increase in 5-HT concentration; p = 0.02) and by L-NAME (p < 0.05). Valve cusps contracted in response to ET-1 (+0.29 ± 0.08% change in load per 10-fold increase in ET-1 concentration; p = 0.02), which was inhibited by cytochalasin-B.
Conclusions: These data highlight the role of the endothelium in regulating the mechanical properties of aortic valve cusps and underline the importance of valve cellular integrity for optimal valve function.
Key Words: aortic valve • mechanical properties • endothelium • nitric oxide
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Abbreviations and Acronyms
| | CyB = cytochalasin B | | ET = endothelin | | 5-HT = 5-hydroxytryptamine | | L-NAME = N-nitro-L-arginine-methyl-ester | | NO = nitric oxide | | SMA = smooth muscle alpha-actin |
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The role of endothelial cells in preventing platelet aggregation, inflammation, and smooth muscle cell contraction and proliferation in the vascular system has been well documented. Endothelial dysfunction is an early occurrence in the cascade of events leading to atherosclerosis. The physiological role of heart valve endothelium has not been adequately defined. Recent studies have indicated that heart valve endothelial cells have specific cellular and molecular characteristics that are not shared with endothelium elsewhere in the vasculature (1,2). While vascular endothelium plays a fundamental role in regulating blood vessel tone, the functional role of endothelium-derived mediators on the mechanical properties of cusp tissue remains unknown. This could be important as the valve is subjected to high mechanical forces at each cardiac cycle, ranging from compression to stretch and shear stress, and is constantly required to adapt to varying hemodynamic conditions. Appropriate adaptations of the mechanical properties of aortic valves are relevant to its function. They can affect stress distribution on the valve cusps (3), improve leaflet coaptation during diastole (3), and optimize the instantaneous movements of the valve, which could influence flow dynamics, coronary perfusion, and ventricular function (4). Therefore, elucidating the regulation of aortic valve mechanical properties will be relevant to understanding the short- and long-term function of heart valves.
We theorize that under physiological loading conditions, aortic valve tissue stiffness (elastic modulus) varies in response to endothelium-derived vasoactive agents, and that the valve endothelium regulates leaflet tone and valve function. The aims of the study are to evaluate the endothelium-dependent changes in aortic valve mechanical properties.
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Methods
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Tissue isolation.
Porcine hearts (18 to 24 months old) were obtained from a commercial slaughterhouse (Cheale Meats, Essex, United Kingdom). Aortic cusps were placed in ice-cold Kreb's buffer (in mM: KCl 2.7, NaCl 136.9, CaCl2 2.5, NaHCO3 11.9, NaH2PO4 0.4, MgCl2 2.5, dextrose 11.1, Na2ethylenediamine tetra-acetic acid 0.04) and used within 12 h of sacrifice. A square-shaped cutter was used to isolate identical 10 x 10 mm sections from the "belly" of each cusp in a radial-circumferential direction. Sections were randomly allocated to 4 different groups according to the vasoactive agent used: 5-hydroxytryptamine (5-HT) (10–8 to 10–5 M; n = 6; Sigma-Aldrich, Dorset, United Kingdom), endothelin-1 (ET-1) (10–10 to 10–8 M; n = 6; Sigma-Aldrich), sodium nitroprusside (10–7 to 10–5 M; n = 6; Sigma-Aldrich), and control (n = 4). Serotonin (5-HT) was chosen for several reasons. It has been shown to induce nitric oxide (NO) release from coronary endothelium (5). Additionally, 5-HT receptors have previously been characterized in aortic valve tissue (6), and importantly 5-HT is also capable of inducing valve contractions (7) by inducing release of intracellular calcium in valve interstitial cells (8). Therefore, it is perfectly suited to evaluate relaxation or contraction responses in aortic valves. ET-1 is an endothelium-derived peptide that has previously been localized in valvular endothelial cells (9). Each tissue section was only exposed to a single vasoactive agent to avoid interactions between various agents.
Role of the endothelium.
The role of the endothelium was evaluated by pharmacological and mechanical means. Cusps were exposed to N-nitro-L-arginine-methyl ester (L-NAME) (10–1 M; Sigma-Aldrich), a nitric oxide synthase inhibitor, to evaluate the role of endothelial-derived NO responses (n = 4). In other cusps, the endothelium was mechanically removed using a cell scraper, before exposing the tissue to vasoactive agents (denuded group, n = 4).
Contribution of interstitial contractile responses.
To evaluate the contribution of interstitial cell contractions to changes in cusp mechanical properties, specimens (n = 4) were incubated for 4 h in cytochalasin B (CyB) (2 µmol/l; Sigma-Aldrich), an actin depolymerizing agent, before testing (10).
Immunohistochemistry and scanning electron microscopy.
Valve cusps were fixed in formalin and stained for von Willebrand factor (endothelial cells) and smooth muscle alpha-actin (SMA [smooth muscle cells]) to evaluate the integrity and distribution of the endothelium before and after testing. Intact and denuded specimens were fixed in glutaraldehyde, and scanning electron microscopy was performed to assess cellular composition.
Experimental protocol.
Stainless steel springs were carefully threaded through each side of the cusp to preserve endothelial integrity. Four square-shaped dots were placed on the central region of the specimen. The specimens were mounted in a biaxial micromechanical testing device (Bose Electroforce, Eden Prairie, Minnesota) in a Kreb's bath maintained at 37°C and continuously gassed with 95% O2/5% CO2 (Fig. 1A). The pH of the solution was held constant between 7.35 and 7.45. Strain gauge force transducers were mounted along both axes to monitor the changes of force in the radial and circumferential directions. A single camera 2-dimensional measuring system was used to track the movement of the dots, measuring real-time strains on the specimen. All tests were conducted equibiaxially.
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Figure 1 Biaxial Micromechanical System and Normal Load-Strain Curves
(A) Porcine aortic cusp mounted to biaxial micromechanical testing device. The 4 dots are tracked using a 60 frames/s charge-coupled device camera to determine the strains on the specimen. (B) Normal load-strain curve of an aortic cusp in the radial direction (blue diamonds) and circumferential direction (red squares) showing the initial toe region, transition region, and linear region. The linear region was used as a measure of the elastic modulus of the valve.
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Tissue stiffness (modulus) measurements.
Aortic valve physiological load was estimated using Laplace's law for cylinders (T = Pr), where T is mean valve membrane tension, P is transvalvular pressure, and r is radius. A transvalvular pressure of 80 mm Hg (106.7 kPa) was used. Each sample was first preconditioned to this level at a frequency of 0.1 Hz for 20 cycles to allow the load-strain response of the valve to become repeatable. Three cycles of load-strain measurements were then recorded for radial and circumferential axes (baseline group). The sample was then stretched to 55 N/m on both axes and allowed to relax 10 min with the strain held constant. The tissue was then challenged with increasing concentrations of vasoactive agents while load measurements were performed every 0.1 s. Three more cycles of load versus strain were recorded (treatment group). Each sample served as its own control and was used only once. Load data were plotted against areal strain (calculated using the radial and circumferential strains obtained above) for baseline and treated groups. Percentage change in cusp stiffness in response to different agents is reported.
To account for the coupling between the radial and circumferential axes in determining valve mechanical properties, areal strain was used as a measure of cusp tissue stiffness. Areal strains are calculated by incorporating simultaneously measured radial and circumferential strains, as detailed in the Appendix. The gradient of the linear portion of the load-areal strain curve was used as a measure of the stiffness/modulus of the valve leaflet (Fig. 1B). This is the region where the collagen fibers are fully uncrimped and the load-bearing components of the valve cusp are recruited. In this manuscript, the terms stiffness, modulus, and elastic modulus are used interchangeably.
Data and statistical analysis.
Data are expressed as mean ± SE. For each valve specimen, the percentage change in load from pre-test values was calculated in the radial and circumferential axes, as a function of the vasoactive agent concentration. Mixed effect models were used to account for the correlation between measurements in the radial and circumferential directions and between repeated measurements in each valve specimen (the MIXED procedure in SAS software, version 9.1, SAS Institute, Cary, North Carolina). Valve percentage changes in load versus control, were tested to assess if any of the mean responses were different from control. If this test was significant, we tested the percentage change at each concentration using a Dunnett correction. To further characterize the concentration-response relationship, more parsimonious models were fitted using linear trend over the log of the concentrations with or without an interaction term, namely, allowing different slopes for the linear trend in radial and circumferential directions. Additionally, 2-sided Student t tests for independent samples were used to compare the changes in elastic modulus between groups. All values of p < 0.05 were considered statistically significant. The statistical analyses were performed using SAS version 9.1 (SAS Institute).
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Results
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Endothelium-dependent changes in aortic valve mechanical properties.
Basal Aortic Valve Elastic Modulus (Stiffness)
Baseline stiffness was evaluated for 4 initial groups with respect to areal strain: control, endothelial denudation, L-NAME alone, and CyB alone (Table 1). Mean baseline stiffness for control aortic cusps was 0.82 ± 0.08 kN/m. The modulus was not affected by endothelial denudation (0.72 ± 0.09 kN/m; p = 0.4 vs. control) nor by the addition of L-NAME (0.66 ± 0.18; p = 0.5 vs. control). Addition of CyB to control cusps significantly reduced the baseline stiffness compared with control (0.36 ± 0.11; p < 0.05 vs. control).
Endothelium Regulation of Changes in Tissue Elastic Modulus
Overall changes in areal strain are presented in Figure 2. Individual variations in the radial and circumferential moduli in response to various stimuli are presented in Table 1. Tissue stiffness was significantly decreased by 5-HT (–25.0 ± 4.0%; p < 0.01 vs. control). However, in endothelium-denuded specimens, addition of 5-HT resulted in a significant increase in cusp areal strain compared with control (+14.7 ± 1.8%; p < 0.05 vs. control). Similarly, when L-NAME was present in the tissue bath, there was a trend toward an increase in cusp areal strain after addition of 5-HT (+17.5 ± 5.3%; p = 0.07 vs. control). Furthermore, the changes in elastic modulus in response to 5-HT in denuded cusps or after addition of L-NAME were each statistically different from the changes after addition of 5-HT alone (both p < 0.01).
ET-1 significantly increased the areal stiffness of aortic valve cusps (+34.4 ± 13.8%; p < 0.05 vs. control). After incubation of the cusps in CyB, addition of ET-1 did not result in a significant difference in cusp stiffness (p = 0.29 vs. control). However, direct comparison of changes after ET-1 versus ET-1 and CyB was statistically significant (p < 0.05).
Endothelial regulation of aortic valve contraction and relaxation responses.
Tissue Relaxation
Endothelium-dependent relaxations
When challenged with 5-HT, the valve specimens relaxed in a concentration-dependent manner (–0.29 ± 0.1% change in load per 10-fold increase in 5-HT concentration; p = 0.03) (Fig. 3A). In the denuded specimens (Fig. 3B), the cusps exhibited significant concentration-dependent contractions to 5-HT in both axes (+0.29 ± 0.06% change in load per 10-fold increase in 5-HT concentration; p = 0.02). Similarly, in the presence of L-NAME, the cusps exhibited a significant concentration-dependent contraction in response to 5-HT in both axes (Fig. 3C).
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Figure 3 Concentration-Response Curves to 5-HT
Concentration-dependent changes in contraction or relaxation (± SE) of porcine aortic valve specimens in response to (A) 5-hydroxytryptamine (5-HT) alone, (B) 5-HT + endothelial denudation, and (C) 5-HT + N-nitro-L-arginine-methyl-ester (L-NAME) in the radial direction (squares) and circumferential direction (diamonds). *Dunnett-adjusted p < 0.05 versus control. **Dunnett-adjusted p < 0.01 versus control.
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Endothelium-independent relaxations
Valve specimens relaxed in a concentration-dependent fashion when challenged with sodium nitroprusside confirming the capacity of contractile cells to relax (p = 0.02; data not shown).
Tissue Contractility in Response to ET-1
Aortic valve cusps significantly contracted in response to increasing concentrations of ET-1 (+0.29 ± 0.08% change in load per 10-fold increase in ET-1 concentration; p = 0.02) (Fig. 4A). When valve specimens were pre-incubated in CyB, the contractile response to ET-1 was mitigated (Fig. 4B).
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Figure 4 Concentration-Response Curves to ET-1
Concentration-dependent changes in contraction or relaxation (± SE) of aortic cusps in response to (A) endothelin (ET)-1 and (B) ET-1 + cytochalasin B (CyB) in the radial (squares) and circumferential (diamonds) axes. *Dunnet-adjusted p < 0.05 versus control. **Dunnett-adjusted p < 0.01 versus control.
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Valve cellular composition.
Endothelial Integrity
Figure 5A is an electron micrograph showing preservation of the endothelial cells on the valve surface, and Figure 5B shows positive von Willebrand factor staining. Figure 5C is a representative electron micrograph of a denuded specimen showing absence of endothelial cells and normal subendothelial structure.
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Figure 5 Cellular Integrity
(A) Scanning electron micrograph of aortic cusp before biaxial testing, showing preserved endothelial monolayer. (Note: areas of apparent endothelial denudation are due to air drying of specimens with hexamethyldisilazane. Both ridges of the endothelium are juxtaposable.) (B) Histological section (original magnification x40) showing preservation of the endothelial cells after biaxial testing by positive von Willebrand factor staining (brown). (C) Scanning electron micrograph of a denuded porcine aortic valve showing absence of endothelium and normal subendothelial structure. (D) Representative immunohistochemical analysis of smooth muscle  -actin (SMA) expression before stimulation with endothelin (ET)-1 (original magnification x20). (E) SMA expression after stimulation with ET-1 (original magnification x20) showing no changes in overall SMA expression.
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Analysis of Contractile Phenotype
All cusps showed intact SMA expression before testing. No differences in expression or distribution of SMA were observed after stretching, including in cusps presenting contractile responses (Fig. 5D).
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Discussion
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The major findings from this study are, under physiological loading conditions, the endothelium significantly modulates the mechanical properties of aortic valve cusps. These mechanisms could regulate valve and flow dynamics and possibly allow long-term optimal cusp function in its unique mechanical environment.
The endothelium releases a number of bioactive substances in vivo, notably NO and ET-1. Whereas NO is a smooth muscle relaxant, ET-1 causes a potent contraction of smooth muscle cells through stimulation of specific receptor subtypes. Both ETA and ETB receptors have previously been shown to be functional in aortic valve tissue (9). Use of exogenous ET-1 serves to demonstrate the effect of this peptide if released by the endothelium. In this study, 5-HT was used to stimulate NO release by endothelial cells (5). There are multiple known 5-HT receptor subtypes, a number of which have been characterized in aortic valve tissue (6,11). Under the present experimental conditions, it appears that the endothelium does not release basal NO or keep the tissue in a constitutively relaxed tone, as evidenced by the absence of change in valve stiffness after pharmacological inhibition or mechanical denudation of the endothelium. However, after addition of 5-HT under these conditions, valve responses are reversed. The observed increase in stiffness suggests that 5-HT acts on normal endothelium by inducing a release of NO. By inhibiting NO synthesis, the direct effect of 5-HT on valve interstitial cells is left unopposed and results in valve contraction. Thus, the endothelium plays a major role in modulating the response to 5-HT. This can affect crucial elements of valve function and possibly impact the long-term durability of the cusps. Changes in stiffness can optimize stress distribution across the surface of the cusps, thus avoiding microtraumatic lesions to the valves. Also, changes in elastic modulus can maintain an adequate surface of coaptation despite high diastolic pressures by reducing the cusps' compliance. These are important elements, considering the high mechanical forces that the thin aortic cusps are exposed to at every cycle and because of the frequent variations in its humoural and hemodynamic environment. The half-life of NO in the cellular environment is very short; it may thus have an impact on the beat-to-beat regulation of valve biomechanics. Conversely, ET-1, which has much longer action duration, is more likely to affect the general tone of the valve, allowing it to adapt to its environment over a longer period of time.
The changes in the mechanical properties of valve tissue in response to different mediators can be explained by the presence of myofibroblasts and smooth muscle cells in the valve interstitial space, which contain SMA and other sarcomeric proteins (12). These cells respond to vasoactive agents, including 5-HT and ET-1, by increases in intracellular calcium (8). The contractile capacity of aortic cusps and valve interstitial cells to both these mediators and others has previously been described and is thought to mediate the responses observed in the present experiments (7,13–15). In addition to these changes, we have documented concurrent changes in tissue stiffness when the cusps are held at physiological tension. Importantly, changes in elastic modulus consistently correlated with relaxation or contraction responses of the tissue to the various mediators. Although as expected, the magnitude of the contraction/relaxation responses was quite small, the magnitude of changes in elastic modulus was relatively high. This disproportionate response is partly explained by the mathematical definition of tissue stress ( ), which is inversely proportional to the surface area (A) of the tissue (  1/A). Therefore, small changes in the leaflet area (A) through contraction or relaxation cause much larger changes in stresses and consequently in the elastic modulus.
The presence of cross-talk between endothelial and interstitial cell populations as suggested by this study is probably part of a unique integrated system linking the function of the different components of the valve (4,16). Recent evidence shows that valvular endothelial cells are phenotypically and genotypically different from vascular endothelial cells (1,2) and react differently in response to shear stress (1,2). Activation of valve endothelial cells after mechanical stimulation involves mechanotransduction pathways that can induce synthesis of bioactive substances, expression of focal adhesion molecules, and reorganization of endothelial cell alignment (17). Potential mechanical cues involve stretch, shear stress or compression. Butcher et al. (1) have described the importance of the Rho-kinase and calpain pathways in valve endothelial cells in response to shear stress. Unlike vascular endothelial cells, the phosphoinositide-3 kinase pathway was not necessary, thus highlighting the unique structure and function of valve endothelial cells. Further studies are required to evaluate the pathways involved in response to other mechanical stimuli as well as the differences between endothelial cells from the aortic and ventricular sides of the valve (18).
Changes in valve endothelial-dependent mechanical responses are likely to be significant in the pathophysiology of valve disease. It has been shown that endothelial damage or dysfunction—through the loss of anti-inflammatory and antioxidant mediators—is an early occurrence in the cascade of events leading to structural valve disease (19). Our data add a new element to this paradigm: after pharmacological inhibition or mechanical denudation of the endothelium, changes in valve elastic modulus in response to endogenous mediators is significantly altered. That may expose specific regions of the cusps to higher than normal stresses, increasing the risk of local microtrauma, which in turn leads to further endothelial damage and structural degeneration. Thus, a loss of the capacity of aortic valves to adapt to their mechanical environment can significantly compound and possibly accelerate structural valve degeneration.
Aside from its role as an endothelium-dependent agonist, 5-HT has been directly linked to valve pathologies, namely, the carcinoid syndrome (20) and secondary to certain appetite suppressants (21,22). The valvular pathologies reported in both these entities have focused on the structural changes in the valves. Although these are the major determinants of disease manifestation, no studies have evaluated the mechanical properties of these valves and their responses to various agonists.
Study limitations.
This is an ex vivo study and remains an approximation to actual in vivo loading conditions. However, while actual values of the mechanical parameters may differ from in vivo values, ex vivo mechanical testing is extremely sensitive to small changes between test groups, and is ideal for a study of this nature. To be consistent in data analysis, we have chosen to use the linear portion of the load-strain curve to calculate elastic modulus for both directions. Due to the inherent differences in tissue architecture between the radial and circumferential directions, using an equibiaxial stretching protocol meant that strains sometimes exceeded the physiological range in the radial direction. The acute experimental setup does not account for the long-term trophic effects of these agents or their ability to modulate the extracellular matrix (15), and it would therefore be difficult to speculate on them. Finally, in vivo aortic cusps are exposed to various mechanical stimuli, including shear stress. Although this model did not address the role of shear stress on valve endothelium, we expect the effect of shear to compound the observed results.
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Conclusions
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We have demonstrated that the mechanical properties of aortic valve cusps are actively regulated through endothelial-dependent pathways. The valve endothelium exerts its effects by modulating aortic valve cusp relaxation and contraction to different mediators, but most importantly by regulating the changes in the stiffness of the cusps. Changes in valve mechanical properties, especially its elastic modulus, could explain the unique ability of aortic valves to withstand severe mechanical stresses during each cardiac cycle. It also suggests possible pathophysiological mechanisms involved in valve disease. Further work is required to assess the effects of these changes on the function of the valves in an in vivo setting, as well as their effect on the long-term durability of heart valves after endothelial injury. It is hoped that these findings will help the understanding of valve physiology and establish a blueprint of adaptive responses of valve mechanical properties for future tissue-engineered heart valves.
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Appendix
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Areal Strain Calculation
Areal (Green's) strain: EA =  ([ RC]2 – 1), where:
R = (2ER + 1)
C = (2EC + 1)
R = radial stretch ratio
C = circumferential stretch ratio
EC = radial (Green's) strain
ER = circumferential (Green's) strain
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Footnotes
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Dr. El-Hamamsy is supported by a Research Fellowship Award from the Canadian Institutes of Health Research (CIHR MFE-83809) and by the Magdi Yacoub Institute. Dr. Balachandran is supported by the National Science Foundation through the ERC program at Georgia Institute of Technology under award number EEC-9731643. Drs. El-Hamamsy and Balachandran contributed equally to this work.
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References
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Y. Matsumoto, V. Adams, S. Jacob, N. Mangner, G. Schuler, and A. Linke
Regular Exercise Training Prevents Aortic Valve Disease in Low-Density Lipoprotein-Receptor-Deficient Mice
Circulation,
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A. N. DeMaria, J. J. Bax, O. Ben-Yehuda, G. K. Feld, B. H. Greenberg, J. Hall, M. Hlatky, W. Y.W. Lew, J. A.C. Lima, A. S. Maisel, et al.
Highlights of the Year in JACC 2009
J. Am. Coll. Cardiol.,
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C. A. Simmons
Aortic Valve Mechanics: An Emerging Role for the Endothelium
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April 21, 2009;
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