LFS-13979; No of Pages 6 Life Sciences xxx (2014) xxx–xxx
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Changes in biomechanical properties of the coronary artery wall contribute to maintained contractile responses to endothelin-1 in atherosclerosis Chen Yen Ooi b, Michael P.F. Sutcliffe b, Anthony P. Davenport a, Janet J. Maguire a,⁎ a b
Clinical Pharmacology Unit, University of Cambridge, UK Department of Engineering, University of Cambridge, UK
a r t i c l e
i n f o
Article history: Received 30 October 2013 Accepted 27 March 2014 Available online xxxx Keywords: Arterial stiffness Atherosclerosis Biomechanical properties Endothelin-1 Human coronary artery Vasoconstriction
a b s t r a c t Aims: Our aim was to determine whether alterations in biomechanical properties of human diseased compared to normal coronary artery contribute to changes in artery responsiveness to endothelin-1 in atherosclerosis. Main methods: Concentration–response curves were constructed to endothelin-1 in normal and diseased coronary artery. The passive mechanical properties of arteries were determined using tensile ring tests from which finite element models of passive mechanical properties of both groups were created. Finite element modelling of artery endothelin-1 responses was then performed. Key findings: Maximum responses to endothelin-1 were significantly attenuated in diseased (27 ± 3 mN, n = 55) compared to normal (38 ± 2 mN, n = 68) artery, although this remained over 70% of control. There was no difference in potency (pD2 control = 8.03 ± 0.06; pD2 diseased = 7.98 ± 0.06). Finite element modelling of tensile ring tests resulted in hyperelastic shear modulus μ = 2004 ± 410 Pa and hardening exponent α = 22.8 ± 2.2 for normal wall and μ = 2464 ± 1075 Pa and α = 38.3 ± 6.7 for plaque tissue and distensibility of diseased vessels was decreased. Finite element modelling of active properties of both groups resulted in higher muscle contractile strain (represented by thermal reactivity) of the atherosclerotic artery model than the normal artery model. The models suggest that a change in muscle response to endothelin-1 occurs in atherosclerotic artery to increase its distensibility towards that seen in normal artery. Significance: Our data suggest that an adaptation occurs in medial smooth muscle of atherosclerotic coronary artery to maintain distensibility of the vessel wall in the presence of endothelin-1. This may contribute to the vasospastic effect of locally increased endothelin-1 production that is reported in this condition. © 2014 Elsevier Inc. All rights reserved.
Introduction The endothelin (ET) system is up-regulated in human coronary atherosclerosis with elevated levels of ET-1 detected in plasma (Lerman et al. 1991), correlating to the extent of disease (Salomone et al., 1996) and with increased ET-1 expression within the atherosclerotic plaque (Zeiher et al., 1995; Bacon et al., 1996). Interestingly, in response to this increased synthesis of ET peptide in human atherosclerosis detected in vitro (Maguire and Davenport., 1998a; Ihling et al., 2001) and in vivo (Böhm et al., 2002) we find no corresponding increase in density of ET receptors in the medial smooth muscle layer of coronary artery and indeed there is a profound down-regulation of ET receptor
⁎ Corresponding author at: Clinical Pharmacology Unit, Level 6 Addenbrooke's Centre for Clinical Investigation, Box 110 Addenbrooke's Hospital, Cambridge CB2 0QQ, UK. Tel.: +44 1223 762579; fax: +44 1223 762564. E-mail address: [email protected] (J.J. Maguire).
expression in the non-contractile intimal smooth muscle layer (Bacon et al., 1996; Maguire and Davenport, 2000; Katugampola et al., 2002). Histological analysis of diseased coronary artery has demonstrated marked atrophy of the contractile medial smooth muscle in the remodelled arterial wall that is predicted to contribute to plaque instability (Burke et al., 2002). The mechanical properties of atherosclerotic artery have been studied both in vitro (Beattie et al., 1998; Baldewsing et al., 2008) and in vivo (van Popele et al., 2001; Baldewsing et al., 2008; Wykretowicz et al., 2009), where the diseased artery has been shown to be stiffer than healthy artery in clinical studies. Holzapfel and Ogden (2010) have provided a review of the studies on the theoretical modelling of arterial smooth muscle function. These include the study of Yang et al. (2003) on a model integrating the electro- and mechano-chemical functions of smooth muscle cell, as well as that of Zulliger et al. (2004) on a pseudo-strain energy function describing the arterial mechanical properties with the coupling of collagen, elastin, and smooth muscle active properties. Despite the well-modelled smooth muscle functions
http://dx.doi.org/10.1016/j.lfs.2014.03.027 0024-3205/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Ooi CY, et al, Changes in biomechanical properties of the coronary artery wall contribute to maintained contractile responses to endothelin-1 in ..., Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.03.027
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in these studies, the changes of arterial active properties with atherosclerotic disease remain unclear. Our hypothesis was that the contractile responses to ET-1 in isolated rings of human atherosclerotic coronary artery should be markedly attenuated compared to histologically normal vessels. This should be a consequence of the relative reduction in the amount of contractile smooth muscle present, compounded by a decrease in arterial distensibility resulting from gross changes in the vessel structure, particularly intimal thickening, increased fibrosis and the presence of lipid laden and calcified plaque (Stary et al., 1994, 1995). Our aim was to compare contractile responses to ET-1 in human normal and atherosclerotic coronary artery in vitro and to develop finite element (FE) models to compare the passive and active mechanical properties of these vessels in health and disease. We first performed mechanical tests and FE modelling to obtain the passive mechanical properties (stretch and distensibility in response to mechanical loading in the absence of chemically mediated smooth muscle cell activation) of both the normal and diseased arteries. We then used these determined passive properties in further FE modelling of the arterial active response to ET-1 to understand how atherosclerosis-induced changes in arterial structure and mechanical properties may affect this active response to ET-1. Unexpectedly we find that compared to histologically normal coronary artery, in vitro contractile responses to ET-1 are well maintained in atherosclerotic vessels with FE modelling demonstrating the capacity of medial smooth muscle cells in diseased arteries to develop elevated contractile strains in response to ET-1 compared to normal arteries.
equilibration, cumulative concentration–response curves (CRC) were then constructed to ET-1 (10− 10–10−6 M) and experiments were completed by addition of 100 mM KCl to determine maximum possible contractile response for each tissue. ET-1 responses were expressed as force developed (mN) or % terminal KCl response. Data were analysed using the non-linear iterative curve-fitting programme GraphPad Prism (GraphPad Software Inc., La Jolla, USA) to determine values of pD2 (−log10 of the EC50 (the concentration of ET-1 that produces 50% of the maximum ET-1 response)) and maximum response (EMAX). Passive tensile ring tests The passive mechanical properties of normal and atherosclerotic human coronary arteries were determined using tensile ring tests. The arterial samples were snap frozen to maintain structural integrity, stored at -70 °C and defrosted before use. The defrosted samples were expected to have negligible smooth muscle cell activation induced by stretching. The artery rings were set up as above, uniaxial loadings were applied to the rings and the reaction forces and arterial deformations in response to mechanical stretch were recorded in both normal (n = 5) and atherosclerotic (n = 6) arterial rings. The mechanical stretch λ of the ring was calculated as: λ¼
2L C
ð1Þ
where L is the distance between the two wires that were used to stretch the arterial ring, and C is the inner circumference of the ring without loading, which was measured from the histology image of the artery.
Materials Human tissue Anonymised human coronary artery samples were used in this study with local ethical approval (REC 05/Q0104/142). Samples were obtained from Papworth Hospital Research Tissue Bank (Cambridgeshire 1 Research Ethics Committee reference 08/H0304/ 56) and were collected with written informed patient consent. Histologically normal arteries (no visible evidence of atherosclerosis) were from 68 patients transplanted for cardiomyopathies (Dec and Fuster, 1994) and atherosclerotic artery samples with evidence of coronary artery disease (CAD, visible plaque present) were from 55 patients transplanted for ischaemic heart disease. n-Values refer to the number of patients from whom tissue was obtained. Artery samples for the contractile functional experiments were transferred to the laboratory in Krebs solution and used within 12 h of retrieval. ET-1 was purchased from the Peptide Institute (Osaka, Japan). Stock solutions (10− 4 M) were made up in 0.1% acetic acid and stored at −70 °C. Krebs solution was of the following composition: (mM) NaCl 90, KCl 5, MgS04.7H2O 0.5, Na2HPO4 1, NaHCO3 45, CaCl2 2.25, glucose 10, Na pyruvate 5, fumaric acid 5, and L-glutamic acid 5. Methods Human coronary artery functional assay Experiments were carried out as previously described (Maguire, 2002). Briefly, 4 mm rings of histologically normal (n = 68) or atherosclerotic (n = 55) endothelium-denuded human coronary artery were set up for isometric force recordings in 5 ml organ baths containing oxygenated Krebs solution at 37 °C. For diseased arteries all 4 mm segments contained visible atherosclerotic plaque, although plaque size was variable between samples. Following an active force normalisation procedure to determine optimum resting force, successful removal of the endothelium was confirmed by lack of reversal of the contractile response to 10 μM phenylephrine by 1 μM acetylcholine. After 30 minute
Finite element models of the passive mechanical properties of normal and atherosclerotic coronary artery For each normal artery sample the FE model was created using Abaqus (Dassault Systèmes, Vélizy-Villacoublay, France), comprising a single circular ring with diameter and wall thickness estimated from the vessel histology (Fig. 1A). The models were assigned with first order Ogden hyperelastic properties with shear modulus μ and hardening exponent α (Abaqus Theory Manual), assuming incompressible material. The model reaction forces were matched with those of the corresponding tensile ring tests using an error minimisation approach with MATLAB (MathWorks, Natick, USA) to determine the optimum μ and α that provide the best matching. Mean values of μ and α of the normal artery models were then calculated and expressed as mean ± sem. A similar approach was applied to the atherosclerotic artery models, with an additional inner vessel wall layer included to represent plaque tissue (Fig. 1B). For atherosclerotic artery the wall outer layer was assigned with the same mean values of μ and α determined for normal artery and the additional inner plaque layer was assigned with different optimum values of μ and α determined by matching the modelled force–stretch results with those of the passive tensile ring tests for the diseased artery samples. In order to compare the overall stiffness of the normal and atherosclerotic arteries, each of the FE models was loaded with internal physiological pressures of 80 mm Hg and 120 mm Hg. The distensibility D of each arterial sample was determined from the geometry changes of the model using Eq. (2): D¼
A120 −A80 A80 ðP 120 −P 80 Þ
ð2Þ
where A80, A120, P80, and P120 are the lumen area A and pressure P at 80 mm Hg and 120 mm Hg, respectively. The stiffness of the arteries was examined by this normalised D (i.e. change of lumen area normalised by the initial area) rather than the non-normalised D to prevent the
Please cite this article as: Ooi CY, et al, Changes in biomechanical properties of the coronary artery wall contribute to maintained contractile responses to endothelin-1 in ..., Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.03.027
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A
Force
Force
B
3
Arterial wall
Arterial wall Plaque Fixed Fixed
C
Pre-stretch
D Adventitia
Adventitia
Contraction Fixed Fixed
Media Media
Plaque
Fig. 1. Finite element models of rings of human normal and diseased coronary artery. (A) Normal and (B) atherosclerotic artery models for the tensile ring tests. (C) Normal and (D) atherosclerotic artery models for the modelling of the contractile response to ET-1.
influence of the significant geometry differences between the normal and diseased groups. Finite element modelling of artery ET-1 responses Two-dimensional FE models that had the geometries of idealised normal and atherosclerotic artery rings were created. The normal artery model contained two separated regions of adventitia and media (Fig. 1C), with diameter and wall thickness estimated from the arterial rings used in the tensile ring experiments. Both of these regions were assigned with the previously derived mean values of hyperelastic μ and α determined for normal artery wall. The medial layer was assigned with an additional thermal expansion property to model the muscle force generation in response to addition of ET-1 under isometric conditions, where applied temperature in the model represents the applied concentration of ET-1 used in the contraction experiments. The thermal expansion property was modelled with an error function (erf) (Eq. (3)) using a user subroutine to generate the reaction forces that match the ET-1 contraction experiments: ε ¼ P 1 ½erf ðP 2 T Þ
P3
ð3Þ
ε and T are the strain and applied temperature, respectively, and P1, P2, and P3 are three parameters that were modified to generate the forces that match the ET-1 contraction experiments. The idea of this modelling approach is to use the thermal modelling capability within the Abaqus FE software to represent directly the contractile effect of ET-1. The relationship between thermal strain and temperature represented in Eq. (3) should be interpreted directly as the effect of ET-1 concentration T on the muscle contraction strain ε. The value of P1 governs the maximum contractile response in the muscle, while P2 and P3 control the shape of the curve representing the relationship between contractile strain and ET-1 concentration. P2
determines the shift of the curve to left or right and P3 determines the slope of the curve. The model was first applied with a pre-stretch load (25 mN) that corresponded to the mean basal force determined by the normalisation procedure in the contraction studies in normal arteries. Thermal loadings of the same magnitudes of the administered ET-1 concentrations were then applied. The results of the model were compared to those of the ET-1 contraction experiment and an error minimisation approach was performed using MATLAB to change the three parameters of Eq. (3) with multiple iterations until a minimum average difference was achieved. The model of the atherosclerotic artery ring was then created, which contained an additional layer of plaque (Fig. 1D), the size of which was estimated from the atherosclerotic samples used in the tensile ring experiments. The adventitia and media were again modelled with the same properties as those of the normal artery model. The plaque region was assigned with hyperelastic μ and α determined from the atherosclerotic artery tensile tests. A pre-stretch load (36 mN) was applied, derived from the normalised basal force determined in the contraction study in diseased arteries, and the same thermal loadings were applied to the diseased artery model. The reaction forces were then matched with the contractile responses to ET-1 using a MATLAB error minimisation approach by modifying the P1, P2, and P3 thermal parameters of the atherosclerotic model. The atherosclerosis-induced changes of arterial contraction under physiological condition were then examined by applying internal pressures of 80 mm Hg and 120 mm Hg to both normal and atherosclerotic models, along with a thermal loading of 3 × 10−7 K (equivalent to 3 × 10−7 M ET-1). The distensibility D of each model was then calculated using Eq. (2), and comparisons were made between the cases of without applying muscle contraction (passive), applying the determined normal muscle tone to both models, and applying diseased muscle tone to the diseased model.
Please cite this article as: Ooi CY, et al, Changes in biomechanical properties of the coronary artery wall contribute to maintained contractile responses to endothelin-1 in ..., Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.03.027
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smooth muscle cells of the diseased arteries generate more force per cell to ET-1 than those of the normal arteries. The distensibility D of these two models under the physiological condition without applying contractile muscle tone (passive) and applying healthy or diseased muscle tones was determined from the modelling of the ET-1 response (Table 1). The diseased model had a lower D than the normal model in the passive condition, as previously obtained in the modelling of the tensile ring tests. Applying the diseased muscle tone significantly increased D for the diseased model, which achieved a similar level as that of the normal model with normal muscle tone.
Results ET-1 mediated contractile responses in normal and atherosclerotic human coronary artery Optimised basal forces were significantly different in normal (25 ± 2 mN) and diseased (36 ± 3 mN) coronary artery. ET-1 contracted normal (pD2 = 8.03 ± 0.06, n = 68) and diseased (pD2 = 7.98 ± 0.06, n = 55) coronary artery with comparable potency. Absolute maximum responses to ET-1 were significantly greater in normal (EMAX 38 ± 2 mN) than in diseased (27 ± 3 mN) artery (Fig. 2A) but when normalised to the terminal KCl response no difference in maximum response to ET-1 in the two groups was obtained (normal, EMAX 78 ± 2% KCl; CAD, EMAX 76 ± 2% KCl) (Fig. 2B), indicating that the reduced response to ET-1 most likely reflected a generalised decrease in contractility of the atherosclerotic tissue rather than an ET-1 specific effect.
Discussion Medial contractile smooth muscle atrophy and proliferation of noncontractile intimal smooth muscle are hallmarks of human atherosclerosis. We hypothesised that these changes would contribute to a marked loss of contractile response to ET-1 in isolated rings of human diseased coronary artery compared to histologically normal artery. However, although we found a significant reduction in the maximum response to ET-1 in diseased artery, the response was still over 70% of that in control arteries. The consequence of this maintained constrictor response for patients may be that in vivo ET-1 produced locally in the diseased vessel wall would produce focal vasospasm resulting in profound reduction in coronary blood flow owing to the already compromised lumen. Variant angina, coronary vasospasm frequently associated with atherosclerosis, can be life threatening but in a recent case has been treated with bosentan implying that ETmediated vasoconstriction is a likely cause at least in some individuals (Krishnan et al., 2010). In human normal and atherosclerotic coronary artery in vitro we have previously demonstrated that contractile responses to ET-1 are mediated via the ETA receptor (Maguire and Davenport, 1998b, 2000). We find no increase in ET receptor density and no up-regulation of ETB receptors in human atherosclerosis (Bacon et al., 1996) to explain this maintained contractile response to ET-1 in disease and therefore we investigated the passive mechanical properties of normal and atherosclerotic coronary arteries using tensile ring tests and FE analysis. Our data indicated that the diseased arteries were stiffer (i.e. exhibited lower distensibility) than normal arteries, in agreement with previous in vivo observations (van Popele et al., 2001; Wykretowicz et al., 2009). Distensibility was chosen as the indicator of arterial stiffness owing to its direct representation of the overall arterial compliance and close correlation with the volume of blood flow through the artery. Elastic modulus, which is commonly used as a representation of stiffness, varies across the plaque and arterial wall of the thickened walled atherosclerotic artery (Beattie et al., 1998; Baldewsing et al., 2008) and is therefore not an appropriate indicator of overall arterial compliance. The distensibility in the passive condition is determined
Passive mechanical properties of normal and atherosclerotic coronary artery Passive tensile ring test data showing the force–stretch relationship for normal and diseased artery samples are shown in Fig. 2C. Matching the force–stretch results of the normal artery FE models with those of the tensile ring tests resulted in mean hyperelastic shear modulus μ = 2004 ± 410 Pa and hardening exponent α = 22.8 ± 2.2 for the vessel wall of normal artery. These values were applied to the wall regions of the atherosclerotic FE models, resulting in values for the plaque region of μ = 2464 ± 1075 Pa and α = 38.3 ± 6.7. The mean distensibility D of the normal and diseased artery samples derived from the FE models were 1.12 ± 0.11 × 10−3 mm Hg−1 and 0.87 ± 0.18 × 10−3 mm Hg−1 respectively, indicating a trend for the normal artery wall to exhibit greater distensibility than the diseased vessels.
Finite element modelling of the ET-1 response in normal and atherosclerotic coronary artery The FE models of the normal and atherosclerotic arteries applied with pre-stretch forces and thermal loadings are shown in Fig. 3A and B. The optimum matching of the reaction forces between the ET-1 experiments and the normal (Fig. 3A) and diseased (Fig. 3B) FE models resulted in parameters of the thermal property of the normal artery model of P1 = − 0.086, P2 = 2.6 × 107 K− 1, and P3 = 0.34, whereas those of the atherosclerotic artery model were P1 = − 0.273, P2 = 3.6 × 107 K− 1, and P3 = 0.20. The negative values of P1 for both models resulted in the contraction of artery rings in response to ET-1. The higher magnitude of P1 required for the atherosclerotic artery model indicated that the maximum strain associated with the drug response was greater in the diseased than in the normal arteries. This suggests that
A
B 30
Response (%KCl)
Response (mN)
40
Normal CAD
20 10 0 -11 -10
-9
-8
-7
log10 ET-1 [M]
-6
C
100 80
Normal CAD
Force (mN)
4
60 40
300
Normal CAD
200
100
20 0 -11 -10
-9
-8
-7
log10 ET-1 [M]
-6
0 1.0
1.1
1.2
1.3
1.4
Stretch
Fig. 2. (A) Contractile responses to ET-1 in histologically normal (●) and diseased (■ CAD) coronary artery expressed in mN force. (B) The same data normalised to the response of 100 mM KCl in each vessel ring. (C) Passive tensile ring test data showing the force–stretch relationship for normal (●) and diseased (■ CAD) coronary artery samples.
Please cite this article as: Ooi CY, et al, Changes in biomechanical properties of the coronary artery wall contribute to maintained contractile responses to endothelin-1 in ..., Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.03.027
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A
5
B
25 mN
Normal 36 mN
Experiment 30 20 10 0 -11 -10 -9
-8
-7
40
Response (mN)
Response (mN)
Atherosclerotic
FE Model
40
FE Model Experiment
30 20 10 0 -11 -10 -9
-6
log10 ET-1 [M]
-8
-7
-6
log10 ET-1 [M]
Fig. 3. Finite element models of (A) normal and (B) atherosclerotic arteries loaded with pre-stretch forces and thermal contractions, with adjacent graphs showing the matching of reaction forces from the ET-1 contractile experiments by the FE models.
by the material properties of the arterial wall, and future studies to determine how changes in distensibility correlate to alterations in the expression of, for example extracellular matrix proteins, would be informative. Indeed, recently the first layer-specific comparative proteomic analyses of human atherosclerotic coronary intima and media have been reported, which indicated changes in the intimal expression of extracellular matrix proteins collagen α-1 (IV) and microfibril-associated glycoprotein 4 and in the markers of contractile phenotype, SM22α and myosin regulatory light 2 (de la Cuesta et al., 2011). Whereas in the media there was evidence for dysregulation of cytoskeleton proteins consistent with the switch of medial smooth muscle cells from contractile to synthetic phenotype, which may also explain the compression and thinning of the medial layer in response to intimal thickening (de la Cuesta et al., 2013). The mechanisms of the arterial responses to ET-1 were demonstrated using FE models, where the diseased artery had similar, or slightly lower, reaction forces when compared with the normal artery. The maintained ability of diseased arteries to contract to ET-1 to a similar extent as normal arteries, despite loss of smooth muscle, may result from an increase of the reactivity of the remaining smooth muscle cells to ET-1, as indicated in the FE modelling where the P1 parameter (thermal reactivity) was higher in the atherosclerotic compared to normal artery model. There may be an underestimate of the diseased model P1, as medial thickness was kept the same in the FE normal and diseased models for ease of comparison rather than reduced in the diseased model to reflect medial atrophy. A limitation of this study is that contractile responses have not been normalised to medial thickness; however, this avoids any ambiguity in identifying the active medial layer in the diseased artery that would introduce uncertainty into the analysis. Therefore we predict that the magnitude of the contractile response P1 for the diseased artery would be even higher if the medial thickness of the atherosclerotic artery model were reduced, while the force response was maintained. Finally the physiological distensibility results of the normal and atherosclerotic models with muscle tones suggest that the diseased artery has an adaptation response which changes muscle reactivity to increase the arterial contraction to applied ET-1, resulting in a distensibility that is closer to that of the normal artery. The mechanisms underlying this observation require further investigation. Table 1 Distensibility D of the normal and diseased atherosclerotic (CAD) models without applying contractile muscle tone (passive) and applying normal or diseased muscle tones determined from the modelling of the ET-1 response. Artery
Normal CAD
D (mm Hg−1) Passive
Normal tone
Diseased tone
1.10 × 10−3 0.67 × 10−3
1.08 × 10−3 0.67 × 10−3
– 1.04 × 10−3
Conclusion This study has identified a maintained active response of human atherosclerotic artery to ET-1 in vitro despite extensive thinning of the media smooth muscle layer. As well as providing comparisons of the mechanical properties of the normal and diseased arteries, the tensile tests and FE modelling demonstrated the potential elevated contractile strains developed by diseased smooth muscle cells in response to ET-1. These results suggest that adaptation mechanisms occur with the pathogenesis of atherosclerosis to maintain the distensibility of the diseased vessel wall in response to vasoconstrictor such as ET-1. If ET-1 production is increased in the atherosclerotic artery then the maintained ability of the diseased artery wall to respond to this peptide may contribute to local vasospasm that is associated with this disease. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by the British Heart Foundation (JJM, PG/09/ 050/27734). We thank the Pulmonary Hypertension Association UK for financial support. This study was supported in part by the NIHR Cambridge Biomedical Research Centre, a project grant from The Tunku Abdul Rahman Centenary Fund, St Catharine's College, University of Cambridge, UK and an Engineering for Clinical Practice Grant, Department of Engineering, University of Cambridge, UK. References Abaqus theory manual. Section 4.6.1. Abaqus 6.9 Documentation Collection, Dassault Systèmes Simulia Corp. 2009. Bacon CR, Cary NRB, Davenport AP. Endothelin peptide and receptors in human atherosclerotic coronary artery and aorta. Circ Res 1996;79:794–801. Baldewsing RA, Danilouchkine MG, Mastik F, Schaar JA, Serruys PW, van der Steen AFW. An inverse method for imaging the local elasticity of atherosclerotic coronary plaques. IEEE Trans Inf Technol Biomed 2008;12:277–89. Beattie D, Xu G, Vito R, Glagov S, Whang WG. Mechanical analysis of heterogeneous, atherosclerotic human aorta. J Biomech Eng 1998;120:602–7. Böhm F, Johansson BL, Hedin U, Alving K, Pernow J. Enhanced vasoconstrictor effect of big endothelin-1 in patients with atherosclerosis: relation to conversion to endothelin-1. Atherosclerosis 2002;160:215–22. Burke AP, Kolodgie FD, Farb A, Weber D, Virmani R. Morphological predictors of arterial remodeling in coronary atherosclerosis. Circulation 2002;105:297–303. de la Cuesta F, Alvarez-Llamas G, Maroto AS, Donado A, Zubiri I, Posada M, et al. A proteomic focus on the alterations occurring at the human atherosclerotic coronary intima. Mol Cell Proteomics 2011;10. [M110.003517]. de la Cuesta F, Zubiri I, Maroto AS, Posada M, Padial LR, Vivanco F, et al. Deregulation of smooth muscle cell cytoskeleton within the human atherosclerotic coronary media layer. J Proteomics 2013;82:155–65. Dec GW, Fuster V. Idiopathic dilated cardiomyopathy. N Engl J Med 1994;331:1564–75. Holzapfel GA, Ogden RW. Constitutive modelling of arteries. Proc R Soc A 2010;466: 1551–97.
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Changes in biomechanical properties of the coronary artery wall contribute to maintained contractile responses to endothelin-1 in atherosclerosis Chen Yen Ooi b, Michael P.F. Sutcliffe b, Anthony P. Davenport a, Janet J. Maguire a,⁎ a b
Clinical Pharmacology Unit, University of Cambridge, UK Department of Engineering, University of Cambridge, UK
a r t i c l e
i n f o
Article history: Received 30 October 2013 Accepted 27 March 2014 Available online xxxx Keywords: Arterial stiffness Atherosclerosis Biomechanical properties Endothelin-1 Human coronary artery Vasoconstriction
a b s t r a c t Aims: Our aim was to determine whether alterations in biomechanical properties of human diseased compared to normal coronary artery contribute to changes in artery responsiveness to endothelin-1 in atherosclerosis. Main methods: Concentration–response curves were constructed to endothelin-1 in normal and diseased coronary artery. The passive mechanical properties of arteries were determined using tensile ring tests from which finite element models of passive mechanical properties of both groups were created. Finite element modelling of artery endothelin-1 responses was then performed. Key findings: Maximum responses to endothelin-1 were significantly attenuated in diseased (27 ± 3 mN, n = 55) compared to normal (38 ± 2 mN, n = 68) artery, although this remained over 70% of control. There was no difference in potency (pD2 control = 8.03 ± 0.06; pD2 diseased = 7.98 ± 0.06). Finite element modelling of tensile ring tests resulted in hyperelastic shear modulus μ = 2004 ± 410 Pa and hardening exponent α = 22.8 ± 2.2 for normal wall and μ = 2464 ± 1075 Pa and α = 38.3 ± 6.7 for plaque tissue and distensibility of diseased vessels was decreased. Finite element modelling of active properties of both groups resulted in higher muscle contractile strain (represented by thermal reactivity) of the atherosclerotic artery model than the normal artery model. The models suggest that a change in muscle response to endothelin-1 occurs in atherosclerotic artery to increase its distensibility towards that seen in normal artery. Significance: Our data suggest that an adaptation occurs in medial smooth muscle of atherosclerotic coronary artery to maintain distensibility of the vessel wall in the presence of endothelin-1. This may contribute to the vasospastic effect of locally increased endothelin-1 production that is reported in this condition. © 2014 Elsevier Inc. All rights reserved.
Introduction The endothelin (ET) system is up-regulated in human coronary atherosclerosis with elevated levels of ET-1 detected in plasma (Lerman et al. 1991), correlating to the extent of disease (Salomone et al., 1996) and with increased ET-1 expression within the atherosclerotic plaque (Zeiher et al., 1995; Bacon et al., 1996). Interestingly, in response to this increased synthesis of ET peptide in human atherosclerosis detected in vitro (Maguire and Davenport., 1998a; Ihling et al., 2001) and in vivo (Böhm et al., 2002) we find no corresponding increase in density of ET receptors in the medial smooth muscle layer of coronary artery and indeed there is a profound down-regulation of ET receptor
⁎ Corresponding author at: Clinical Pharmacology Unit, Level 6 Addenbrooke's Centre for Clinical Investigation, Box 110 Addenbrooke's Hospital, Cambridge CB2 0QQ, UK. Tel.: +44 1223 762579; fax: +44 1223 762564. E-mail address: [email protected] (J.J. Maguire).
expression in the non-contractile intimal smooth muscle layer (Bacon et al., 1996; Maguire and Davenport, 2000; Katugampola et al., 2002). Histological analysis of diseased coronary artery has demonstrated marked atrophy of the contractile medial smooth muscle in the remodelled arterial wall that is predicted to contribute to plaque instability (Burke et al., 2002). The mechanical properties of atherosclerotic artery have been studied both in vitro (Beattie et al., 1998; Baldewsing et al., 2008) and in vivo (van Popele et al., 2001; Baldewsing et al., 2008; Wykretowicz et al., 2009), where the diseased artery has been shown to be stiffer than healthy artery in clinical studies. Holzapfel and Ogden (2010) have provided a review of the studies on the theoretical modelling of arterial smooth muscle function. These include the study of Yang et al. (2003) on a model integrating the electro- and mechano-chemical functions of smooth muscle cell, as well as that of Zulliger et al. (2004) on a pseudo-strain energy function describing the arterial mechanical properties with the coupling of collagen, elastin, and smooth muscle active properties. Despite the well-modelled smooth muscle functions
http://dx.doi.org/10.1016/j.lfs.2014.03.027 0024-3205/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Ooi CY, et al, Changes in biomechanical properties of the coronary artery wall contribute to maintained contractile responses to endothelin-1 in ..., Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.03.027
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in these studies, the changes of arterial active properties with atherosclerotic disease remain unclear. Our hypothesis was that the contractile responses to ET-1 in isolated rings of human atherosclerotic coronary artery should be markedly attenuated compared to histologically normal vessels. This should be a consequence of the relative reduction in the amount of contractile smooth muscle present, compounded by a decrease in arterial distensibility resulting from gross changes in the vessel structure, particularly intimal thickening, increased fibrosis and the presence of lipid laden and calcified plaque (Stary et al., 1994, 1995). Our aim was to compare contractile responses to ET-1 in human normal and atherosclerotic coronary artery in vitro and to develop finite element (FE) models to compare the passive and active mechanical properties of these vessels in health and disease. We first performed mechanical tests and FE modelling to obtain the passive mechanical properties (stretch and distensibility in response to mechanical loading in the absence of chemically mediated smooth muscle cell activation) of both the normal and diseased arteries. We then used these determined passive properties in further FE modelling of the arterial active response to ET-1 to understand how atherosclerosis-induced changes in arterial structure and mechanical properties may affect this active response to ET-1. Unexpectedly we find that compared to histologically normal coronary artery, in vitro contractile responses to ET-1 are well maintained in atherosclerotic vessels with FE modelling demonstrating the capacity of medial smooth muscle cells in diseased arteries to develop elevated contractile strains in response to ET-1 compared to normal arteries.
equilibration, cumulative concentration–response curves (CRC) were then constructed to ET-1 (10− 10–10−6 M) and experiments were completed by addition of 100 mM KCl to determine maximum possible contractile response for each tissue. ET-1 responses were expressed as force developed (mN) or % terminal KCl response. Data were analysed using the non-linear iterative curve-fitting programme GraphPad Prism (GraphPad Software Inc., La Jolla, USA) to determine values of pD2 (−log10 of the EC50 (the concentration of ET-1 that produces 50% of the maximum ET-1 response)) and maximum response (EMAX). Passive tensile ring tests The passive mechanical properties of normal and atherosclerotic human coronary arteries were determined using tensile ring tests. The arterial samples were snap frozen to maintain structural integrity, stored at -70 °C and defrosted before use. The defrosted samples were expected to have negligible smooth muscle cell activation induced by stretching. The artery rings were set up as above, uniaxial loadings were applied to the rings and the reaction forces and arterial deformations in response to mechanical stretch were recorded in both normal (n = 5) and atherosclerotic (n = 6) arterial rings. The mechanical stretch λ of the ring was calculated as: λ¼
2L C
ð1Þ
where L is the distance between the two wires that were used to stretch the arterial ring, and C is the inner circumference of the ring without loading, which was measured from the histology image of the artery.
Materials Human tissue Anonymised human coronary artery samples were used in this study with local ethical approval (REC 05/Q0104/142). Samples were obtained from Papworth Hospital Research Tissue Bank (Cambridgeshire 1 Research Ethics Committee reference 08/H0304/ 56) and were collected with written informed patient consent. Histologically normal arteries (no visible evidence of atherosclerosis) were from 68 patients transplanted for cardiomyopathies (Dec and Fuster, 1994) and atherosclerotic artery samples with evidence of coronary artery disease (CAD, visible plaque present) were from 55 patients transplanted for ischaemic heart disease. n-Values refer to the number of patients from whom tissue was obtained. Artery samples for the contractile functional experiments were transferred to the laboratory in Krebs solution and used within 12 h of retrieval. ET-1 was purchased from the Peptide Institute (Osaka, Japan). Stock solutions (10− 4 M) were made up in 0.1% acetic acid and stored at −70 °C. Krebs solution was of the following composition: (mM) NaCl 90, KCl 5, MgS04.7H2O 0.5, Na2HPO4 1, NaHCO3 45, CaCl2 2.25, glucose 10, Na pyruvate 5, fumaric acid 5, and L-glutamic acid 5. Methods Human coronary artery functional assay Experiments were carried out as previously described (Maguire, 2002). Briefly, 4 mm rings of histologically normal (n = 68) or atherosclerotic (n = 55) endothelium-denuded human coronary artery were set up for isometric force recordings in 5 ml organ baths containing oxygenated Krebs solution at 37 °C. For diseased arteries all 4 mm segments contained visible atherosclerotic plaque, although plaque size was variable between samples. Following an active force normalisation procedure to determine optimum resting force, successful removal of the endothelium was confirmed by lack of reversal of the contractile response to 10 μM phenylephrine by 1 μM acetylcholine. After 30 minute
Finite element models of the passive mechanical properties of normal and atherosclerotic coronary artery For each normal artery sample the FE model was created using Abaqus (Dassault Systèmes, Vélizy-Villacoublay, France), comprising a single circular ring with diameter and wall thickness estimated from the vessel histology (Fig. 1A). The models were assigned with first order Ogden hyperelastic properties with shear modulus μ and hardening exponent α (Abaqus Theory Manual), assuming incompressible material. The model reaction forces were matched with those of the corresponding tensile ring tests using an error minimisation approach with MATLAB (MathWorks, Natick, USA) to determine the optimum μ and α that provide the best matching. Mean values of μ and α of the normal artery models were then calculated and expressed as mean ± sem. A similar approach was applied to the atherosclerotic artery models, with an additional inner vessel wall layer included to represent plaque tissue (Fig. 1B). For atherosclerotic artery the wall outer layer was assigned with the same mean values of μ and α determined for normal artery and the additional inner plaque layer was assigned with different optimum values of μ and α determined by matching the modelled force–stretch results with those of the passive tensile ring tests for the diseased artery samples. In order to compare the overall stiffness of the normal and atherosclerotic arteries, each of the FE models was loaded with internal physiological pressures of 80 mm Hg and 120 mm Hg. The distensibility D of each arterial sample was determined from the geometry changes of the model using Eq. (2): D¼
A120 −A80 A80 ðP 120 −P 80 Þ
ð2Þ
where A80, A120, P80, and P120 are the lumen area A and pressure P at 80 mm Hg and 120 mm Hg, respectively. The stiffness of the arteries was examined by this normalised D (i.e. change of lumen area normalised by the initial area) rather than the non-normalised D to prevent the
Please cite this article as: Ooi CY, et al, Changes in biomechanical properties of the coronary artery wall contribute to maintained contractile responses to endothelin-1 in ..., Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.03.027
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A
Force
Force
B
3
Arterial wall
Arterial wall Plaque Fixed Fixed
C
Pre-stretch
D Adventitia
Adventitia
Contraction Fixed Fixed
Media Media
Plaque
Fig. 1. Finite element models of rings of human normal and diseased coronary artery. (A) Normal and (B) atherosclerotic artery models for the tensile ring tests. (C) Normal and (D) atherosclerotic artery models for the modelling of the contractile response to ET-1.
influence of the significant geometry differences between the normal and diseased groups. Finite element modelling of artery ET-1 responses Two-dimensional FE models that had the geometries of idealised normal and atherosclerotic artery rings were created. The normal artery model contained two separated regions of adventitia and media (Fig. 1C), with diameter and wall thickness estimated from the arterial rings used in the tensile ring experiments. Both of these regions were assigned with the previously derived mean values of hyperelastic μ and α determined for normal artery wall. The medial layer was assigned with an additional thermal expansion property to model the muscle force generation in response to addition of ET-1 under isometric conditions, where applied temperature in the model represents the applied concentration of ET-1 used in the contraction experiments. The thermal expansion property was modelled with an error function (erf) (Eq. (3)) using a user subroutine to generate the reaction forces that match the ET-1 contraction experiments: ε ¼ P 1 ½erf ðP 2 T Þ
P3
ð3Þ
ε and T are the strain and applied temperature, respectively, and P1, P2, and P3 are three parameters that were modified to generate the forces that match the ET-1 contraction experiments. The idea of this modelling approach is to use the thermal modelling capability within the Abaqus FE software to represent directly the contractile effect of ET-1. The relationship between thermal strain and temperature represented in Eq. (3) should be interpreted directly as the effect of ET-1 concentration T on the muscle contraction strain ε. The value of P1 governs the maximum contractile response in the muscle, while P2 and P3 control the shape of the curve representing the relationship between contractile strain and ET-1 concentration. P2
determines the shift of the curve to left or right and P3 determines the slope of the curve. The model was first applied with a pre-stretch load (25 mN) that corresponded to the mean basal force determined by the normalisation procedure in the contraction studies in normal arteries. Thermal loadings of the same magnitudes of the administered ET-1 concentrations were then applied. The results of the model were compared to those of the ET-1 contraction experiment and an error minimisation approach was performed using MATLAB to change the three parameters of Eq. (3) with multiple iterations until a minimum average difference was achieved. The model of the atherosclerotic artery ring was then created, which contained an additional layer of plaque (Fig. 1D), the size of which was estimated from the atherosclerotic samples used in the tensile ring experiments. The adventitia and media were again modelled with the same properties as those of the normal artery model. The plaque region was assigned with hyperelastic μ and α determined from the atherosclerotic artery tensile tests. A pre-stretch load (36 mN) was applied, derived from the normalised basal force determined in the contraction study in diseased arteries, and the same thermal loadings were applied to the diseased artery model. The reaction forces were then matched with the contractile responses to ET-1 using a MATLAB error minimisation approach by modifying the P1, P2, and P3 thermal parameters of the atherosclerotic model. The atherosclerosis-induced changes of arterial contraction under physiological condition were then examined by applying internal pressures of 80 mm Hg and 120 mm Hg to both normal and atherosclerotic models, along with a thermal loading of 3 × 10−7 K (equivalent to 3 × 10−7 M ET-1). The distensibility D of each model was then calculated using Eq. (2), and comparisons were made between the cases of without applying muscle contraction (passive), applying the determined normal muscle tone to both models, and applying diseased muscle tone to the diseased model.
Please cite this article as: Ooi CY, et al, Changes in biomechanical properties of the coronary artery wall contribute to maintained contractile responses to endothelin-1 in ..., Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.03.027
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smooth muscle cells of the diseased arteries generate more force per cell to ET-1 than those of the normal arteries. The distensibility D of these two models under the physiological condition without applying contractile muscle tone (passive) and applying healthy or diseased muscle tones was determined from the modelling of the ET-1 response (Table 1). The diseased model had a lower D than the normal model in the passive condition, as previously obtained in the modelling of the tensile ring tests. Applying the diseased muscle tone significantly increased D for the diseased model, which achieved a similar level as that of the normal model with normal muscle tone.
Results ET-1 mediated contractile responses in normal and atherosclerotic human coronary artery Optimised basal forces were significantly different in normal (25 ± 2 mN) and diseased (36 ± 3 mN) coronary artery. ET-1 contracted normal (pD2 = 8.03 ± 0.06, n = 68) and diseased (pD2 = 7.98 ± 0.06, n = 55) coronary artery with comparable potency. Absolute maximum responses to ET-1 were significantly greater in normal (EMAX 38 ± 2 mN) than in diseased (27 ± 3 mN) artery (Fig. 2A) but when normalised to the terminal KCl response no difference in maximum response to ET-1 in the two groups was obtained (normal, EMAX 78 ± 2% KCl; CAD, EMAX 76 ± 2% KCl) (Fig. 2B), indicating that the reduced response to ET-1 most likely reflected a generalised decrease in contractility of the atherosclerotic tissue rather than an ET-1 specific effect.
Discussion Medial contractile smooth muscle atrophy and proliferation of noncontractile intimal smooth muscle are hallmarks of human atherosclerosis. We hypothesised that these changes would contribute to a marked loss of contractile response to ET-1 in isolated rings of human diseased coronary artery compared to histologically normal artery. However, although we found a significant reduction in the maximum response to ET-1 in diseased artery, the response was still over 70% of that in control arteries. The consequence of this maintained constrictor response for patients may be that in vivo ET-1 produced locally in the diseased vessel wall would produce focal vasospasm resulting in profound reduction in coronary blood flow owing to the already compromised lumen. Variant angina, coronary vasospasm frequently associated with atherosclerosis, can be life threatening but in a recent case has been treated with bosentan implying that ETmediated vasoconstriction is a likely cause at least in some individuals (Krishnan et al., 2010). In human normal and atherosclerotic coronary artery in vitro we have previously demonstrated that contractile responses to ET-1 are mediated via the ETA receptor (Maguire and Davenport, 1998b, 2000). We find no increase in ET receptor density and no up-regulation of ETB receptors in human atherosclerosis (Bacon et al., 1996) to explain this maintained contractile response to ET-1 in disease and therefore we investigated the passive mechanical properties of normal and atherosclerotic coronary arteries using tensile ring tests and FE analysis. Our data indicated that the diseased arteries were stiffer (i.e. exhibited lower distensibility) than normal arteries, in agreement with previous in vivo observations (van Popele et al., 2001; Wykretowicz et al., 2009). Distensibility was chosen as the indicator of arterial stiffness owing to its direct representation of the overall arterial compliance and close correlation with the volume of blood flow through the artery. Elastic modulus, which is commonly used as a representation of stiffness, varies across the plaque and arterial wall of the thickened walled atherosclerotic artery (Beattie et al., 1998; Baldewsing et al., 2008) and is therefore not an appropriate indicator of overall arterial compliance. The distensibility in the passive condition is determined
Passive mechanical properties of normal and atherosclerotic coronary artery Passive tensile ring test data showing the force–stretch relationship for normal and diseased artery samples are shown in Fig. 2C. Matching the force–stretch results of the normal artery FE models with those of the tensile ring tests resulted in mean hyperelastic shear modulus μ = 2004 ± 410 Pa and hardening exponent α = 22.8 ± 2.2 for the vessel wall of normal artery. These values were applied to the wall regions of the atherosclerotic FE models, resulting in values for the plaque region of μ = 2464 ± 1075 Pa and α = 38.3 ± 6.7. The mean distensibility D of the normal and diseased artery samples derived from the FE models were 1.12 ± 0.11 × 10−3 mm Hg−1 and 0.87 ± 0.18 × 10−3 mm Hg−1 respectively, indicating a trend for the normal artery wall to exhibit greater distensibility than the diseased vessels.
Finite element modelling of the ET-1 response in normal and atherosclerotic coronary artery The FE models of the normal and atherosclerotic arteries applied with pre-stretch forces and thermal loadings are shown in Fig. 3A and B. The optimum matching of the reaction forces between the ET-1 experiments and the normal (Fig. 3A) and diseased (Fig. 3B) FE models resulted in parameters of the thermal property of the normal artery model of P1 = − 0.086, P2 = 2.6 × 107 K− 1, and P3 = 0.34, whereas those of the atherosclerotic artery model were P1 = − 0.273, P2 = 3.6 × 107 K− 1, and P3 = 0.20. The negative values of P1 for both models resulted in the contraction of artery rings in response to ET-1. The higher magnitude of P1 required for the atherosclerotic artery model indicated that the maximum strain associated with the drug response was greater in the diseased than in the normal arteries. This suggests that
A
B 30
Response (%KCl)
Response (mN)
40
Normal CAD
20 10 0 -11 -10
-9
-8
-7
log10 ET-1 [M]
-6
C
100 80
Normal CAD
Force (mN)
4
60 40
300
Normal CAD
200
100
20 0 -11 -10
-9
-8
-7
log10 ET-1 [M]
-6
0 1.0
1.1
1.2
1.3
1.4
Stretch
Fig. 2. (A) Contractile responses to ET-1 in histologically normal (●) and diseased (■ CAD) coronary artery expressed in mN force. (B) The same data normalised to the response of 100 mM KCl in each vessel ring. (C) Passive tensile ring test data showing the force–stretch relationship for normal (●) and diseased (■ CAD) coronary artery samples.
Please cite this article as: Ooi CY, et al, Changes in biomechanical properties of the coronary artery wall contribute to maintained contractile responses to endothelin-1 in ..., Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.03.027
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A
5
B
25 mN
Normal 36 mN
Experiment 30 20 10 0 -11 -10 -9
-8
-7
40
Response (mN)
Response (mN)
Atherosclerotic
FE Model
40
FE Model Experiment
30 20 10 0 -11 -10 -9
-6
log10 ET-1 [M]
-8
-7
-6
log10 ET-1 [M]
Fig. 3. Finite element models of (A) normal and (B) atherosclerotic arteries loaded with pre-stretch forces and thermal contractions, with adjacent graphs showing the matching of reaction forces from the ET-1 contractile experiments by the FE models.
by the material properties of the arterial wall, and future studies to determine how changes in distensibility correlate to alterations in the expression of, for example extracellular matrix proteins, would be informative. Indeed, recently the first layer-specific comparative proteomic analyses of human atherosclerotic coronary intima and media have been reported, which indicated changes in the intimal expression of extracellular matrix proteins collagen α-1 (IV) and microfibril-associated glycoprotein 4 and in the markers of contractile phenotype, SM22α and myosin regulatory light 2 (de la Cuesta et al., 2011). Whereas in the media there was evidence for dysregulation of cytoskeleton proteins consistent with the switch of medial smooth muscle cells from contractile to synthetic phenotype, which may also explain the compression and thinning of the medial layer in response to intimal thickening (de la Cuesta et al., 2013). The mechanisms of the arterial responses to ET-1 were demonstrated using FE models, where the diseased artery had similar, or slightly lower, reaction forces when compared with the normal artery. The maintained ability of diseased arteries to contract to ET-1 to a similar extent as normal arteries, despite loss of smooth muscle, may result from an increase of the reactivity of the remaining smooth muscle cells to ET-1, as indicated in the FE modelling where the P1 parameter (thermal reactivity) was higher in the atherosclerotic compared to normal artery model. There may be an underestimate of the diseased model P1, as medial thickness was kept the same in the FE normal and diseased models for ease of comparison rather than reduced in the diseased model to reflect medial atrophy. A limitation of this study is that contractile responses have not been normalised to medial thickness; however, this avoids any ambiguity in identifying the active medial layer in the diseased artery that would introduce uncertainty into the analysis. Therefore we predict that the magnitude of the contractile response P1 for the diseased artery would be even higher if the medial thickness of the atherosclerotic artery model were reduced, while the force response was maintained. Finally the physiological distensibility results of the normal and atherosclerotic models with muscle tones suggest that the diseased artery has an adaptation response which changes muscle reactivity to increase the arterial contraction to applied ET-1, resulting in a distensibility that is closer to that of the normal artery. The mechanisms underlying this observation require further investigation. Table 1 Distensibility D of the normal and diseased atherosclerotic (CAD) models without applying contractile muscle tone (passive) and applying normal or diseased muscle tones determined from the modelling of the ET-1 response. Artery
Normal CAD
D (mm Hg−1) Passive
Normal tone
Diseased tone
1.10 × 10−3 0.67 × 10−3
1.08 × 10−3 0.67 × 10−3
– 1.04 × 10−3
Conclusion This study has identified a maintained active response of human atherosclerotic artery to ET-1 in vitro despite extensive thinning of the media smooth muscle layer. As well as providing comparisons of the mechanical properties of the normal and diseased arteries, the tensile tests and FE modelling demonstrated the potential elevated contractile strains developed by diseased smooth muscle cells in response to ET-1. These results suggest that adaptation mechanisms occur with the pathogenesis of atherosclerosis to maintain the distensibility of the diseased vessel wall in response to vasoconstrictor such as ET-1. If ET-1 production is increased in the atherosclerotic artery then the maintained ability of the diseased artery wall to respond to this peptide may contribute to local vasospasm that is associated with this disease. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by the British Heart Foundation (JJM, PG/09/ 050/27734). We thank the Pulmonary Hypertension Association UK for financial support. This study was supported in part by the NIHR Cambridge Biomedical Research Centre, a project grant from The Tunku Abdul Rahman Centenary Fund, St Catharine's College, University of Cambridge, UK and an Engineering for Clinical Practice Grant, Department of Engineering, University of Cambridge, UK. References Abaqus theory manual. Section 4.6.1. Abaqus 6.9 Documentation Collection, Dassault Systèmes Simulia Corp. 2009. Bacon CR, Cary NRB, Davenport AP. Endothelin peptide and receptors in human atherosclerotic coronary artery and aorta. Circ Res 1996;79:794–801. Baldewsing RA, Danilouchkine MG, Mastik F, Schaar JA, Serruys PW, van der Steen AFW. An inverse method for imaging the local elasticity of atherosclerotic coronary plaques. IEEE Trans Inf Technol Biomed 2008;12:277–89. Beattie D, Xu G, Vito R, Glagov S, Whang WG. Mechanical analysis of heterogeneous, atherosclerotic human aorta. J Biomech Eng 1998;120:602–7. Böhm F, Johansson BL, Hedin U, Alving K, Pernow J. Enhanced vasoconstrictor effect of big endothelin-1 in patients with atherosclerosis: relation to conversion to endothelin-1. Atherosclerosis 2002;160:215–22. Burke AP, Kolodgie FD, Farb A, Weber D, Virmani R. Morphological predictors of arterial remodeling in coronary atherosclerosis. Circulation 2002;105:297–303. de la Cuesta F, Alvarez-Llamas G, Maroto AS, Donado A, Zubiri I, Posada M, et al. A proteomic focus on the alterations occurring at the human atherosclerotic coronary intima. Mol Cell Proteomics 2011;10. [M110.003517]. de la Cuesta F, Zubiri I, Maroto AS, Posada M, Padial LR, Vivanco F, et al. Deregulation of smooth muscle cell cytoskeleton within the human atherosclerotic coronary media layer. J Proteomics 2013;82:155–65. Dec GW, Fuster V. Idiopathic dilated cardiomyopathy. N Engl J Med 1994;331:1564–75. Holzapfel GA, Ogden RW. Constitutive modelling of arteries. Proc R Soc A 2010;466: 1551–97.
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