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The cardiac valve interstitial cell
The International Journal of Biochemistry & Cell Biology 35 (2003) 113–118
Cells in focus
The cardiac valve interstitial cell Patricia M. Taylor∗ , Puspa Batten, Nigel J. Brand, Penny S. Thomas, Magdi H. Yacoub Division of Cardiothoracic Surgery, National Heart and Lung Institute, Faculty of Medicine, Imperial College of Science, Technology and Medicine at Harefield Hospital, Heart Science Centre, Harefield, Middlesex, UB9 6JH, UK Received 31 January 2002; received in revised form 10 June 2002; accepted 19 June 2002
Abstract Cardiac valve interstitial cells (ICs) are a heterogeneous and dynamic population of specific cell types that have many unique characteristics. They are responsible for maintaining the extracellular scaffold that provides the mechanical characteristics vital for sustaining the unique dynamic behaviour of the valve. A number of cellular phenotypes can be distinguished: some are sparsely arranged throughout the valve leaflets, whilst others are arranged in thin bundles. These cells express molecular markers similar to those of skeletal, cardiac and smooth muscle cells (SMCs) and in particular, many ICs express smooth muscle (SM) ␣-actin, a marker of myofibroblasts. In this respect, these cells exhibit a profile unlike skin fibroblasts, which may allude to their role in valve function. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Cardiac valves; Interstitial cells; Myofibroblasts
This article discusses the characteristics and functions of these unique cells. Cell facts • heterogeneous population of specific phenotypes; • characteristics of skeletal, cardiac and SMCs; • express SM ␣-actin; • maintain the extracellular matrix; • unique and specific role in valve function; • modulate immune response.
1. Introduction Cardiac valves perform a complex and sophisticated series of functions over a wide range of haemodynamic conditions [1]. The interstitial cells (ICs) that populate the valve leaflets are thought to play a vital ∗ Corresponding author. Tel.: +44-1895-828889; fax: +44-1895-828900. E-mail address: [email protected] (P.M. Taylor).
role in maintaining their function. The non-cellular component of the cardiac valve consists of a matrix of collagen, elastic fibres, proteoglycans and glycoproteins. ICs lie within the matrix and endothelial cells (ECs), which are continuous with the endocardium of the heart and the endothelium of the sinuses and great vessels, cover the surfaces of the valve leaflets. The extracellular matrix components have been extensively studied, however less attention has been given to the cellular elements and their function. Studies of
1357-2725/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 2 ) 0 0 1 0 0 - 0
114
P.M. Taylor et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 113–118
native valve leaflets demonstrate that the valve matrix is only sparsely populated with cells and it is likely that each phenotype has an important and specific role to perform. Characterisation of the cellular composition of valve leaflets or cells cultured from porcine and human valves has demonstrated the heterogeneity of valve ICs [2].
being replaced by cells that migrate from the surrounding tissues. It has been proposed that fibroblasts differentiate along a terminal differentiation lineage with increasing age [4]. It has also been shown that the number of cells in human valve leaflets decreases with age and is accompanied by a degeneration of collagen fibres [5], which demonstrates the importance of these cells in maintaining a healthy valve matrix.
2. Cell origin and lifespan 3. Cell phenotype Valve leaflets are derived from embryonic mesenchymal outgrowths referred to as the cardiac cushions. The cells that occupy, maintain and modify these structures are descended from endocardial cells that have undergone an epithelial-to-mesenchymal transformation, regulated by the myocardium [3] and cells that have migrated in from extra-cardiac sites during embryonic development. The precise relative contribution of these different sources is unclear. The rate of cellular turnover within the valve leaflet is not known, since cell proliferation in normal valves has not been demonstrated. In addition, it remains to be ascertained whether the resident population divides to produce new cells or whether they are continually
Two cell morphologies have been observed when valve ICs are cultured; small islands of cuboidal cells and spindle shaped elongated cells. Once confluent, the elongated cells exhibit a swirling pattern, characteristic of fibroblasts and start to pile up on each other in layers. In our experience, valve ICs have a slow rate of proliferation even when a variety of growth factors are added, which may reflect an inherent low replication rate. To date, at least three phenotypes of IC have been identified in the mature native valve [2]. Two cellular phenotypes have been demonstrated by electron microscopy and immunocytochemistry both in situ
Fig. 1. Cellular functions of valve interstitial cells. Valve interstitial cells synthesise and secrete cytokines, chemokines, growth factors, extracellular matrix components, matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), which modulate the matrix and also release growth factors sequestered within the matrix. In addition, these cells may have an immunomodulatory role.
P.M. Taylor et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 113–118
and using ICs cultured from porcine valves. One type, the myofibroblast is characterised by prominent stress fibres, associated with smooth muscle ␣-actin expression and is thought to be involved in proliferation and migration. This contractile phenotype rapidly remodels the extracellular matrix. The other subtype, thought to be important in matrix regulation, is characterised by prominent synthetic and secretory organelles associated with expression of prolyl 4-hydroxylase, an enzyme essential for stabilisation of the collagen triple helix, which indicates that the cells are actively synthesising collagen. In addition to these, smooth muscle cells have been identified occurring either singly or arranged in thin bundles. Although few studies have been carried out using human tissue, similar phenotypes have been identified in the native leaflet and in cells cultured from valve leaflets [6,7]. Definitive identification of individual cell type solely by immunocytochemistry is difficult, since molecular markers exclusively specific for fibroblasts, myofibroblasts or SMCs are not available. Komuro [8] proposed that a family of fibroblast-like cells exist which varies its phenotype as an adaptive response to the microenvironment. It is well documented that extracellular matrix and cytokines are important in fibroblast differentiation and function. This ability of fibroblasts to adapt to their environment could be very useful in the development of fully functioning tissue engineered cardiac valves. These cells synthesise collagen, elastin, proteoglycans, fibronectin, growth factors, cytokines, chemokines as well as matrix metalloproteinases and their tissue inhibitors. Matrix metalloproteinases play a vital role in the remodelling and maintenance of the extracellular matrix in health and disease [9] and control the release Table 1 Mean percentage of cells expressing SM ␣-actin in isolates cultured from human cardiac valves plated on glass coverslips Valve type/cell origin
Aortic Pulmonary Mitral Tricuspid
Expression of SM ␣-actin n
Mean (%)
S.D.
14 3 16 18
78 65 66 56
28 41 33 28
Number of isolates (n) examined and S.D. are also given.
115
of growth factors sequestered within the matrix. The cellular functions of valve ICs are depicted in Fig. 1. Whether the same range of phenotypes is present in all of the valve leaflets still remains to be determined. However, results from this laboratory shown in Table 1 demonstrate that cultured human valve ICs have a similar phenotypic profile, with respect to SM ␣-actin expression, whether they are from the outflow valves (aortic and pulmonary) or the atrioventricular valves (mitral and tricuspid).
4. Myofibroblast nature Myofibroblasts [10] are unique mesenchymal cells found in many tissues that have characteristics of both fibroblasts and SMCs. They display a highly plastic and diverse phenotype, depending on tissue origin and whether the tissue is normal or pathological. Common features include expression of both muscle and non-muscle structural and regulatory proteins, contractile properties and secretion of extracellular matrix. It is likely that many of the ICs in the native valve leaflet are myofibroblasts. Evidence to support this is fourfold. First, many of the cells express SM ␣-actin ([2,6,7,11], Fig. 2A and B) and possess other features similar to SMCs. Second, valve ICs express the skeletal muscle-specific regulatory gene myogenin and genes encoding structural components of the cardiac and skeletal contractile apparatus, the sarcomere [12]. Third, the cells synthesise collagen and other matrix proteins [11] and fourth, the cells are able to contract when immobilised within a collagen gel matrix ( Eastwood, personnel communication). The unique characteristics of myofibroblasts may be central to the lifelong durability of cardiac valves. In addition, they could contribute to the capacity of valve tissue to contract in response to vasoactive agents such as endothelin-1 and thromboxane A2 [13]. The expression of both cardiac and skeletal contractile proteins in valve ICs, which include ␣-myosin heavy chain, ␣- and -tropomyosins and various troponin isoforms [12] indicates the potential for some contractile function. This is reminiscent of embryonic and foetal striated muscle development, in which cardiac isoforms of sarcomeric proteins may be expressed in developing skeletal muscle, before being replaced by skeletal isoforms in adult
116
P.M. Taylor et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 113–118
Fig. 2. (A) Photomicrograph of cultured aortic valve ICs plated on glass coverslips: double exposure of cells stained with Oregon green 514 phalloidin which binds to filamentous actin present in all cells and an antibody against SM ␣-actin tagged with Alexa FluorTM 594 goat anti-mouse IgG. Fibroblasts appear green; SM ␣-actin positive myofibroblasts appear yellow. (B) Two-dimensional confocal image of aortic valve interstitial cells cultured for 2 weeks in collagen sponge and stained for SM ␣-actin as described in (A). (C) Scanning electron micrograph of aortic valve interstitial cell on collagen fibre showing long cellular extensions.
muscle, and vice versa in the development of cardiac muscle.
5. Immunogenicity of cardiac valve ICs An interesting interplay between the ICs and the ECs that line the valves has recently been demonstrated [14]. This may provide an insight into the cellular mechanisms involved in valve degeneration. In order to provoke an immune response resulting in full T cell activation, antigen presenting cells must present two signals to the T cell: signal one being a cognate interaction between MHC class II bound with peptide and the T cell receptor and signal two via a co-stimulatory molecule. If only one signal is delivered, a state of anergy or non-responsiveness can occur. Our studies of cell surface phenotypic characterisation of valve endothelial and ICs revealed that similar levels of major histocompatibility antigens (MHC) classes I and II and adhesion/co-stimulatory
(LFA-3, ICAM-1, CD40) molecules were either induced or upregulated following interferon-␥ treatment. These similarities however, did not reflect the T cell responses to valve ECs or ICs. Only the valve ECs and not the valve ICs stimulated a T cell response following interferon-␥ treatment. A two-step tolerance induction protocol revealed that not only did the ICs fail to provoke a T cell response, they also rendered the T cells non-responsive or anergic to donor-specific valve ECs. They were still responsive to third party valve ECs. This induction of anergy was reversed using anti-MHC class II monoclonal antibodies. Furthermore, the presence of the co-stimulatory molecule B7-1 presented on a bystander cell was shown to substitute for the missing co-stimulatory signal, indicating that the valve ICs lacked the ability to provide co-stimulation. These results imply that the non-immunogenic state of valve ICs may provide alternative routes to down-regulate possible immune responses against tissue-engineered valve leaflets.
P.M. Taylor et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 113–118
6. Cell communication The presence of adherens junctions, indicating tight bonding, as well as gap junctions has been reported in both cultured valve ICs and the native valve leaflet. In addition, functionally active communicating junctions have been elegantly demonstrated in cultured valve ICs [2]. Cells in the native leaflet and when cultured and seeded in three-dimensional collagen scaffolds (Fig. 2C) exhibit long interconnecting processes which could be part of such cell–cell communication. It has also been reported that human cardiac valves have distinct patterns of innervation, which may be involved in the control of valve function [1]. Integrins provide dynamic links between cells and extracellular matrix molecules, mediating both adhesion and signal transduction, which control cell proliferation, survival, gene induction, differentiation and cell motility. We have demonstrated that vasoactive agents induce intracellular calcium transients [6], increase collagen synthesis in cultured valve ICs [15] and induce contraction of isolated valve leaflets [13]. These observations indicate the existence of a number of receptor signalling pathways.
7. Role in valve disease It would appear that valve ICs are vital for the maintenance of the matrix. The fact that the decrease with age in the number of cells in human valve leaflets is accompanied by a degeneration of collagen fibres [5] and the accepted view that degeneration of implanted cryopreserved valves occurs largely because there are no viable cells to produce matrix supports this. However, fixed porcine bioprosthetic valves, which contain no viable cells can function for many years. Recently, strategies aimed at encouraging repopulation in vivo of acellularised valves by host cells have been described [16]. Increased production of matrix metalloproteinases and their tissue inhibitors by valve ICs leading to matrix remodelling has been associated with Marfan’s syndrome [9] and other valvular diseases. Recent studies have indicated that valve ICs contribute to matrix destruction and remodelling in myxomatous mitral valve degeneration [17]. These valve ICs have
117
features of activated myofibroblasts and express elevated levels of catabolic enzymes.
8. Response to injury Injury to the ECs covering the valve leaflets may be one of the important initial steps in the pathogenesis of several valve pathological conditions. Studies of organ cultures of valves as well as animal studies have demonstrated that valve ICs play an important role in the repair process [2]. Valve ICs may also have a detrimental role. It has been shown that calcification of valves may be caused by ICs [18].
9. Conclusion It would appear from our studies and those of others that valve ICs are a dynamic population of specific cell types, which communicate with each other and the extracellular matrix through the release of growth factors, integrin interaction, calcium signalling and intracellular tension. Knowledge of these complex mechanisms of cell–cell interaction and cell–matrix interaction is essential in order to understand how these cells contribute to the physiological function of valves and how the valve attempts to re-establish normal structure and function following injury. These interactions are now beginning to be investigated and understood at the molecular level. References [1] M.H. Yacoub, P.J. Kilner, E.J. Birks, M. Misfeld, The aortic outflow and root: a tale of dynamism and crosstalk, Ann. Thorac. Surg. 68 (1999) S37–S43. [2] D.L. Mulholland, A.I. Gotlieb, Cell biology of valvular interstitial cells, Can. J. Cardiol. 12 (1996) 231–236. [3] L.M. Eisenberg, R.R. Markwald, Molecular regulation of atrioventricular valvuloseptal morphogenesis, Circ. Res. 77 (1995) 1–6. [4] K. Bayreuther, H.P. Rodemann, P.I. Francz, K. Maier, Differentiation of fibroblast stem cells, J. Cell. Sci. Suppl. 10 (1988) 115–130. [5] S. Sell, R.E. Scully, Aging changes in the aortic and mitral valves. Histological and biochemical studies, with observations on the pathogenesis of calcific aortic stenosis and calcification of the mitral annulus, Am. J. Pathol. 46 (1965) 345–365.
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[6] P.M. Taylor, S.P. Allen, M.H. Yacoub, Phenotypic and functional characteristization of interstitial cells from human heart valves, pericardium and skin, J. Heart Valve Dis. 9 (2000) 150–158. [7] F. Della Rocca, S. Sartore, D. Guidolin, B. Bertiplaglia, G. Gerosa, D. Casarotto, P. Pauletto, Cell composition of the human pulmonary valve: a comparative study with the aortic valve: the VESALIO Project, Ann. Thorac. Surg. 70 (2000) 1594–1600. [8] T. Komuro, Re-evaluation of fibroblasts and fibroblast-like cells, Anat. Embryol. 182 (1990) 103–112. [9] A.M. Segura, R.E. Luna, K. Horiba, W.G. Stetler-Stevenson, H.A. McAllister Jr, J.T. Willerson, V.J. Ferrans, Immunohistochemistry of matrix metalloproteinases and their inhibitors in thoracic aortic aneurysms and aortic valves of patients with Marfan’s syndrome, Circulation 98 (1998) II331–II338. [10] A. Schmitt-Graff, A. Desmouliere, G. Gabbiani, Heterogeneity of myofibroblast phenotypic features: an example of fibroblastic cell plasticity, Virchows. Arch. 425 (1994) 3–24. [11] R.H. Messier, B.L. Bass, H.M. Aly, J.L. Jones, P.W. Domkowski, R.B. Wallace, R.A. Hopkins, Dual structural and functional phenotypes of the porcine aortic valve interstitial population: characteristics of the leaflet myofibroblast, J. Surg. Res. 57 (1994) 1–21.
[12] A. Roy, N.J. Brand, M.H. Yacoub, Molecular characterization of interstitial cells isolated from human heart valves, J. Heart Valve Dis. 9 (2000) 459–465. [13] A.H. Chester, M. Misfeld, M.H. Yacoub, Receptor mediated contraction of aortic valve leaflets, J. Heart Valve Dis. 9 (2000) 250–255. [14] P. Batten, A.M. McCormack, M.L. Rose, M.H. Yacoub, Valve interstitial cells induce donor-specific T cell anergy, J. Thorac. Cardiovasc. Surg. 122 (2001) 129–135. [15] S. Hafizi, P.M. Taylor, A.H. Chester, S.P. Allen, M.H. Yacoub, Mitogenic and secretory responses of human valve interstitial cells to vasoactive agents, J. Heart Valve Dis. 9 (2000) 454– 458. [16] R.C. Elkins, P.E. Dawson, S. Goldstein, S.P. Walsh, K.S. Black, Decellularized human valve allografts, Ann. Thorac. Surg. 71 (2001) S428–S432. [17] E. Rabkin, M. Aikawa, J.R. Stone, Y. Fukumoto, P. Libby, F.J. Schoen, Activated interstitial myofibroblasts express catabolic enzymes and mediate matrix remodeling in myxomatous heart valves, Circulation 104 (2001) 2525–2532. [18] E.R. Mohler III, M.K. Chawla, A.W. Chang, N. Vyavahare, R.J. Levy, L. Graham, F.H. Gannon, Identification and characterization of calcifying valve cells from human and canine aortic valves, J. Heart Valve Dis. 8 (1999) 254– 260.
Cells in focus
The cardiac valve interstitial cell Patricia M. Taylor∗ , Puspa Batten, Nigel J. Brand, Penny S. Thomas, Magdi H. Yacoub Division of Cardiothoracic Surgery, National Heart and Lung Institute, Faculty of Medicine, Imperial College of Science, Technology and Medicine at Harefield Hospital, Heart Science Centre, Harefield, Middlesex, UB9 6JH, UK Received 31 January 2002; received in revised form 10 June 2002; accepted 19 June 2002
Abstract Cardiac valve interstitial cells (ICs) are a heterogeneous and dynamic population of specific cell types that have many unique characteristics. They are responsible for maintaining the extracellular scaffold that provides the mechanical characteristics vital for sustaining the unique dynamic behaviour of the valve. A number of cellular phenotypes can be distinguished: some are sparsely arranged throughout the valve leaflets, whilst others are arranged in thin bundles. These cells express molecular markers similar to those of skeletal, cardiac and smooth muscle cells (SMCs) and in particular, many ICs express smooth muscle (SM) ␣-actin, a marker of myofibroblasts. In this respect, these cells exhibit a profile unlike skin fibroblasts, which may allude to their role in valve function. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Cardiac valves; Interstitial cells; Myofibroblasts
This article discusses the characteristics and functions of these unique cells. Cell facts • heterogeneous population of specific phenotypes; • characteristics of skeletal, cardiac and SMCs; • express SM ␣-actin; • maintain the extracellular matrix; • unique and specific role in valve function; • modulate immune response.
1. Introduction Cardiac valves perform a complex and sophisticated series of functions over a wide range of haemodynamic conditions [1]. The interstitial cells (ICs) that populate the valve leaflets are thought to play a vital ∗ Corresponding author. Tel.: +44-1895-828889; fax: +44-1895-828900. E-mail address: [email protected] (P.M. Taylor).
role in maintaining their function. The non-cellular component of the cardiac valve consists of a matrix of collagen, elastic fibres, proteoglycans and glycoproteins. ICs lie within the matrix and endothelial cells (ECs), which are continuous with the endocardium of the heart and the endothelium of the sinuses and great vessels, cover the surfaces of the valve leaflets. The extracellular matrix components have been extensively studied, however less attention has been given to the cellular elements and their function. Studies of
1357-2725/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 2 ) 0 0 1 0 0 - 0
114
P.M. Taylor et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 113–118
native valve leaflets demonstrate that the valve matrix is only sparsely populated with cells and it is likely that each phenotype has an important and specific role to perform. Characterisation of the cellular composition of valve leaflets or cells cultured from porcine and human valves has demonstrated the heterogeneity of valve ICs [2].
being replaced by cells that migrate from the surrounding tissues. It has been proposed that fibroblasts differentiate along a terminal differentiation lineage with increasing age [4]. It has also been shown that the number of cells in human valve leaflets decreases with age and is accompanied by a degeneration of collagen fibres [5], which demonstrates the importance of these cells in maintaining a healthy valve matrix.
2. Cell origin and lifespan 3. Cell phenotype Valve leaflets are derived from embryonic mesenchymal outgrowths referred to as the cardiac cushions. The cells that occupy, maintain and modify these structures are descended from endocardial cells that have undergone an epithelial-to-mesenchymal transformation, regulated by the myocardium [3] and cells that have migrated in from extra-cardiac sites during embryonic development. The precise relative contribution of these different sources is unclear. The rate of cellular turnover within the valve leaflet is not known, since cell proliferation in normal valves has not been demonstrated. In addition, it remains to be ascertained whether the resident population divides to produce new cells or whether they are continually
Two cell morphologies have been observed when valve ICs are cultured; small islands of cuboidal cells and spindle shaped elongated cells. Once confluent, the elongated cells exhibit a swirling pattern, characteristic of fibroblasts and start to pile up on each other in layers. In our experience, valve ICs have a slow rate of proliferation even when a variety of growth factors are added, which may reflect an inherent low replication rate. To date, at least three phenotypes of IC have been identified in the mature native valve [2]. Two cellular phenotypes have been demonstrated by electron microscopy and immunocytochemistry both in situ
Fig. 1. Cellular functions of valve interstitial cells. Valve interstitial cells synthesise and secrete cytokines, chemokines, growth factors, extracellular matrix components, matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), which modulate the matrix and also release growth factors sequestered within the matrix. In addition, these cells may have an immunomodulatory role.
P.M. Taylor et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 113–118
and using ICs cultured from porcine valves. One type, the myofibroblast is characterised by prominent stress fibres, associated with smooth muscle ␣-actin expression and is thought to be involved in proliferation and migration. This contractile phenotype rapidly remodels the extracellular matrix. The other subtype, thought to be important in matrix regulation, is characterised by prominent synthetic and secretory organelles associated with expression of prolyl 4-hydroxylase, an enzyme essential for stabilisation of the collagen triple helix, which indicates that the cells are actively synthesising collagen. In addition to these, smooth muscle cells have been identified occurring either singly or arranged in thin bundles. Although few studies have been carried out using human tissue, similar phenotypes have been identified in the native leaflet and in cells cultured from valve leaflets [6,7]. Definitive identification of individual cell type solely by immunocytochemistry is difficult, since molecular markers exclusively specific for fibroblasts, myofibroblasts or SMCs are not available. Komuro [8] proposed that a family of fibroblast-like cells exist which varies its phenotype as an adaptive response to the microenvironment. It is well documented that extracellular matrix and cytokines are important in fibroblast differentiation and function. This ability of fibroblasts to adapt to their environment could be very useful in the development of fully functioning tissue engineered cardiac valves. These cells synthesise collagen, elastin, proteoglycans, fibronectin, growth factors, cytokines, chemokines as well as matrix metalloproteinases and their tissue inhibitors. Matrix metalloproteinases play a vital role in the remodelling and maintenance of the extracellular matrix in health and disease [9] and control the release Table 1 Mean percentage of cells expressing SM ␣-actin in isolates cultured from human cardiac valves plated on glass coverslips Valve type/cell origin
Aortic Pulmonary Mitral Tricuspid
Expression of SM ␣-actin n
Mean (%)
S.D.
14 3 16 18
78 65 66 56
28 41 33 28
Number of isolates (n) examined and S.D. are also given.
115
of growth factors sequestered within the matrix. The cellular functions of valve ICs are depicted in Fig. 1. Whether the same range of phenotypes is present in all of the valve leaflets still remains to be determined. However, results from this laboratory shown in Table 1 demonstrate that cultured human valve ICs have a similar phenotypic profile, with respect to SM ␣-actin expression, whether they are from the outflow valves (aortic and pulmonary) or the atrioventricular valves (mitral and tricuspid).
4. Myofibroblast nature Myofibroblasts [10] are unique mesenchymal cells found in many tissues that have characteristics of both fibroblasts and SMCs. They display a highly plastic and diverse phenotype, depending on tissue origin and whether the tissue is normal or pathological. Common features include expression of both muscle and non-muscle structural and regulatory proteins, contractile properties and secretion of extracellular matrix. It is likely that many of the ICs in the native valve leaflet are myofibroblasts. Evidence to support this is fourfold. First, many of the cells express SM ␣-actin ([2,6,7,11], Fig. 2A and B) and possess other features similar to SMCs. Second, valve ICs express the skeletal muscle-specific regulatory gene myogenin and genes encoding structural components of the cardiac and skeletal contractile apparatus, the sarcomere [12]. Third, the cells synthesise collagen and other matrix proteins [11] and fourth, the cells are able to contract when immobilised within a collagen gel matrix ( Eastwood, personnel communication). The unique characteristics of myofibroblasts may be central to the lifelong durability of cardiac valves. In addition, they could contribute to the capacity of valve tissue to contract in response to vasoactive agents such as endothelin-1 and thromboxane A2 [13]. The expression of both cardiac and skeletal contractile proteins in valve ICs, which include ␣-myosin heavy chain, ␣- and -tropomyosins and various troponin isoforms [12] indicates the potential for some contractile function. This is reminiscent of embryonic and foetal striated muscle development, in which cardiac isoforms of sarcomeric proteins may be expressed in developing skeletal muscle, before being replaced by skeletal isoforms in adult
116
P.M. Taylor et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 113–118
Fig. 2. (A) Photomicrograph of cultured aortic valve ICs plated on glass coverslips: double exposure of cells stained with Oregon green 514 phalloidin which binds to filamentous actin present in all cells and an antibody against SM ␣-actin tagged with Alexa FluorTM 594 goat anti-mouse IgG. Fibroblasts appear green; SM ␣-actin positive myofibroblasts appear yellow. (B) Two-dimensional confocal image of aortic valve interstitial cells cultured for 2 weeks in collagen sponge and stained for SM ␣-actin as described in (A). (C) Scanning electron micrograph of aortic valve interstitial cell on collagen fibre showing long cellular extensions.
muscle, and vice versa in the development of cardiac muscle.
5. Immunogenicity of cardiac valve ICs An interesting interplay between the ICs and the ECs that line the valves has recently been demonstrated [14]. This may provide an insight into the cellular mechanisms involved in valve degeneration. In order to provoke an immune response resulting in full T cell activation, antigen presenting cells must present two signals to the T cell: signal one being a cognate interaction between MHC class II bound with peptide and the T cell receptor and signal two via a co-stimulatory molecule. If only one signal is delivered, a state of anergy or non-responsiveness can occur. Our studies of cell surface phenotypic characterisation of valve endothelial and ICs revealed that similar levels of major histocompatibility antigens (MHC) classes I and II and adhesion/co-stimulatory
(LFA-3, ICAM-1, CD40) molecules were either induced or upregulated following interferon-␥ treatment. These similarities however, did not reflect the T cell responses to valve ECs or ICs. Only the valve ECs and not the valve ICs stimulated a T cell response following interferon-␥ treatment. A two-step tolerance induction protocol revealed that not only did the ICs fail to provoke a T cell response, they also rendered the T cells non-responsive or anergic to donor-specific valve ECs. They were still responsive to third party valve ECs. This induction of anergy was reversed using anti-MHC class II monoclonal antibodies. Furthermore, the presence of the co-stimulatory molecule B7-1 presented on a bystander cell was shown to substitute for the missing co-stimulatory signal, indicating that the valve ICs lacked the ability to provide co-stimulation. These results imply that the non-immunogenic state of valve ICs may provide alternative routes to down-regulate possible immune responses against tissue-engineered valve leaflets.
P.M. Taylor et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 113–118
6. Cell communication The presence of adherens junctions, indicating tight bonding, as well as gap junctions has been reported in both cultured valve ICs and the native valve leaflet. In addition, functionally active communicating junctions have been elegantly demonstrated in cultured valve ICs [2]. Cells in the native leaflet and when cultured and seeded in three-dimensional collagen scaffolds (Fig. 2C) exhibit long interconnecting processes which could be part of such cell–cell communication. It has also been reported that human cardiac valves have distinct patterns of innervation, which may be involved in the control of valve function [1]. Integrins provide dynamic links between cells and extracellular matrix molecules, mediating both adhesion and signal transduction, which control cell proliferation, survival, gene induction, differentiation and cell motility. We have demonstrated that vasoactive agents induce intracellular calcium transients [6], increase collagen synthesis in cultured valve ICs [15] and induce contraction of isolated valve leaflets [13]. These observations indicate the existence of a number of receptor signalling pathways.
7. Role in valve disease It would appear that valve ICs are vital for the maintenance of the matrix. The fact that the decrease with age in the number of cells in human valve leaflets is accompanied by a degeneration of collagen fibres [5] and the accepted view that degeneration of implanted cryopreserved valves occurs largely because there are no viable cells to produce matrix supports this. However, fixed porcine bioprosthetic valves, which contain no viable cells can function for many years. Recently, strategies aimed at encouraging repopulation in vivo of acellularised valves by host cells have been described [16]. Increased production of matrix metalloproteinases and their tissue inhibitors by valve ICs leading to matrix remodelling has been associated with Marfan’s syndrome [9] and other valvular diseases. Recent studies have indicated that valve ICs contribute to matrix destruction and remodelling in myxomatous mitral valve degeneration [17]. These valve ICs have
117
features of activated myofibroblasts and express elevated levels of catabolic enzymes.
8. Response to injury Injury to the ECs covering the valve leaflets may be one of the important initial steps in the pathogenesis of several valve pathological conditions. Studies of organ cultures of valves as well as animal studies have demonstrated that valve ICs play an important role in the repair process [2]. Valve ICs may also have a detrimental role. It has been shown that calcification of valves may be caused by ICs [18].
9. Conclusion It would appear from our studies and those of others that valve ICs are a dynamic population of specific cell types, which communicate with each other and the extracellular matrix through the release of growth factors, integrin interaction, calcium signalling and intracellular tension. Knowledge of these complex mechanisms of cell–cell interaction and cell–matrix interaction is essential in order to understand how these cells contribute to the physiological function of valves and how the valve attempts to re-establish normal structure and function following injury. These interactions are now beginning to be investigated and understood at the molecular level. References [1] M.H. Yacoub, P.J. Kilner, E.J. Birks, M. Misfeld, The aortic outflow and root: a tale of dynamism and crosstalk, Ann. Thorac. Surg. 68 (1999) S37–S43. [2] D.L. Mulholland, A.I. Gotlieb, Cell biology of valvular interstitial cells, Can. J. Cardiol. 12 (1996) 231–236. [3] L.M. Eisenberg, R.R. Markwald, Molecular regulation of atrioventricular valvuloseptal morphogenesis, Circ. Res. 77 (1995) 1–6. [4] K. Bayreuther, H.P. Rodemann, P.I. Francz, K. Maier, Differentiation of fibroblast stem cells, J. Cell. Sci. Suppl. 10 (1988) 115–130. [5] S. Sell, R.E. Scully, Aging changes in the aortic and mitral valves. Histological and biochemical studies, with observations on the pathogenesis of calcific aortic stenosis and calcification of the mitral annulus, Am. J. Pathol. 46 (1965) 345–365.
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