International Journal of Cardiology 176 (2014) 1252–1254
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
Increased expression of cystatin C and transforming growth factor β-1 in calcific aortic valves Ertan Yetkin a,⁎, Vadim Tchaikovski a, Nevzat Erdil b, Sadet Alan c, Johannes Waltenberger a a b c
Department of Cardiology, University Hospital Maastricht, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, Maastricht, The Netherlands Inonu University School of Medicine, Department of Cardiovascular Surgery, Malatya, Turkey Inonu University School of Medicine, Department of Pathology, Malatya, Turkey
a r t i c l e
i n f o
Article history: Received 25 June 2014 Accepted 27 July 2014 Available online 4 August 2014 Keywords: Cystatin C Growth factor Transforming growth factor Aortic valve Calcification aortic stenosis
Calcific aortic stenosis (AS) is a chronic and progressive disease. Until recently, the concept of calcific aortic valve disease as a degenerative and unmodifiable process basically induced by long-lasting mechanical stress was generally accepted [1] Studies have determined that mature lamellar bone formation occurs in calcified human aortic valves [2,3]. Yetkin and Waltenberger have reviewed potential molecular and cellular mechanisms of aortic stenosis in terms of extracellular matrix remodeling, angiogenesis, and osteoblast-like bone formation in aortic valve tissue [4]. Transforming growth factor beta-1 (TGF-β1) has been reported to play a role in the calcification of interstitial cells of aortic valves via promoting apoptosis [5]. TGF-β1 is a cytokine that has been demonstrated to promote calcification in human aortic valve interstitial cells [3] Cystatin-C is a cysteine protease inhibitor that was previously reported to be expressed in mature osteoblasts [6]. A potential link between Cys C and TGF-β1 has previously been reported in vascular smooth muscle cells [7]. Since the calcification process in human aortic valves resembles the mature lamellar bone formation, it is reasonable to expect the potential role of both molecules in the pathogenesis of calcification of human aortic valves. Accordingly, we aimed to evaluate the expression of TGF-β1 and Cys C in normal (non-calcified) and calcified human aortic valves. ⁎ Corresponding author at: Middle East Hospital, Mersin, Turkey.
http://dx.doi.org/10.1016/j.ijcard.2014.07.199 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.
Leaflets of tricuspid aortic valves were obtained from patients undergoing surgical aortic valve replacement because of calcific aortic stenosis (n = 6), from autopsy and organ donors (n = 4). Surgical specimens were obtained from 6 adults with an average age of 65 years. Aortic valve morphology was evaluated on hematoxylin and eosin stained sections. Cystatin C immunostaining: After deparaffinization process and blocking the endogenous peroxidase activity, sections were subjected to microwave treatment. Afterwards, sections were incubated for 90 min with polyclonal rabbit anti-human cystatin C antibody (1:1000, DAKO Cytomation, Glostrup; Denmark). After washing in TBS, biotinylated swine anti-rabbit IgG (1:150 Dako) was applied for 30 min as secondary antibody. After incubation with an alkaline phosphatase-coupled avidin– biotin complex (ABC complex, DAKO Cytomation, Glostrup; Denmark), both antibodies (TGF-β1 and cystatin C) were visualized with an alkaline substrate kit (Vector SK-5100, Vector Laboratories, Inc., UK). Parallel sections were stained with mouse monoclonal antibodies against TGF-β1 (1:500, Abcam). For the mouse monoclonal antibodies, biotinylated rabbit anti-mouse IgG (1:20) for 30 min was used as the secondary antibody, whereas biotinylated swine anti-rabbit IgG (1:200, Dako) was used as the secondary antibody for the polyclonal antibody. Sections were counterstained with hematoxylin and mounted with coverslips. In negative controls, incubation with primary antibody was omitted. Sections of immunostained specimens were evaluated by three independent observers using a semiquantitative scoring system: negative (−); focal, weak staining (+); present, strong staining (++); and very strong, widespread staining (+++). Western blot analysis of cystatin C and TGF-β1 expression: Crossslices of frozen aortic valves were homogenized on liquid nitrogen. After serial lysis and centrifugation process, supernatants containing 15 μg (cystatin C) or 100 μg (TGF-β1) of protein were heated in β-mercaptoethanol-containing sample buffer at 96 °C for 5 min and resolved on 20% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by a transfer onto nitrocellulose membranes (Amersham, Braunschweig, Germany). Subsequently, membranes were blocked with 5% semi-dry milk solution and incubated with antibody against cystatin C (DAKO Cytomation, Glostrup; Denmark) at a dilution of 1:3000 or TGF-β (Santa Cruz Biotechnology Inc., Santa Cruz,
E. Yetkin et al. / International Journal of Cardiology 176 (2014) 1252–1254
California; USA). The images were captured by scanning with LAS documentation system (Fuji Photo Film Co., Japan). Immunohistochemistry studies of six specimens of calcified aortic cusps revealed that both Cys C staining and TGF-β1 staining were strongly positive at calcified and non-calcified sites in both extracellular and cellular compartments, while those of normal aortic valves were largely negative for TGF-β1. Only the endothelial layer and fibrosa part of the normal aortic valve showed a mild staining for Cys C and TGF-β1. The pattern of Cys C and TGF-β1 immunostaining showed a close similarity, i.e., colocalization, regarding valvular structures such as endothelium and fibrosa, calcified and chondroid areas (Fig. 1), and capillarized and inflammatory areas. In normal human aortic valves, pathologic findings such as calcification, chondroid tissue, inflammatory area and capillary formation were not observed. Western blot analysis confirmed the increased expression of cystatin C in calcified aortic valve tissue compared to normal aortic valve tissue. In accordance with cystatin C expression, increased expression of TGF-β1 was detected in calcified aortic valve tissue compared to normal valve tissue. We have demonstrated that expression of TGF-β1 and cystatin C is significantly enhanced in calcified aortic human valves compared to normal human aortic valves. Additionally expression of TGF-β1 and cystatin C showed similar staining patterns in endothelium, extracellular matrix and inflammatory areas. Rajamannan et al. [8] have demonstrated that calcification in human aortic valve leaflets has similar features to that of osteoblastogenesis during skeletal bone formation. TGF-β1 is a cytokine that has been demonstrated to promote the calcification in human aortic valves [3]. TGF-β1 is characteristically present within calcified stenotic aortic cusps and mediates the calcification process of aortic valve interstitial cells in culture [5]. Similarly, the presence of TGF-β1 has also been shown in carcinoid plaques of the heart valves [9,10]. Cys C as a cysteine protease inhibitor was previously reported to be expressed in mature osteoblasts [6]. Additionally, Cys C has been found to inhibit bone resorption [11–13]. It has been shown that the maximum expression of Cys C appears to coincide with early
1253
events in osteoblast differentiation [14]. Recently, Helske et al. have demonstrated increased expression of elastolytic cathepsins, namely cathepsins K, S, and V and their inhibitor Cys C in stenotic human aortic valves [15]. Since we have demonstrated that expression of both TGF-β1 and Cys C is increased in calcified aortic valves, these molecules are likely to play a role in the calcification process of non-rheumatic aortic stenosis. It has been shown that TGF-β1 clearly regulates Cys C expression vascular smooth muscle [7]. A strong positive regulation of the Cys C gene by TGF-β1 in serum-free mouse embryonic cells has also been reported [16]. In conclusion, we have demonstrated that Cys C and TGF-β1 expression is significantly increased in non-rheumatic calcified aortic valves compared to normal aortic valve tissue. Both molecules have similar expression patterns. However, the mechanisms contributing to the interaction between Cys C and TGF-β1 in calcifying human aortic valve remain to be elucidated. Inhibiting the possible interaction between these two molecules could potentially enable us to prevent the progression of aortic valve calcification at an early stage of the disease.
Conflict of interest The authors report no relationships that could be construed as a conflict of interest.
Acknowledgment We would like to thank Anique Janssen for her technical and skilful assistance during the immunostaining procedure. The study was supported in part by an unrestricted grant from Medtronic BV. The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology.
Fig. 1. Similar expression pattern of Cys C and TGF-β1 in calcified aortic valve tissue. Immunohistochemical detection of Cys C in the calcifying (A1) and endothelial and subendothelial (A2) areas. Immunohistochemical detection of TGF-β1 in the calcifying (B1) and endothelial and subendothelial (B2) areas (primary magnification ×100).
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E. Yetkin et al. / International Journal of Cardiology 176 (2014) 1252–1254
References [1] Rosenhek R, Binder T, Porenta G, et al. Predictors of outcome in severe, asymptomatic aortic stenosis. N Engl J Med 2000;343:611–7. [2] O'Brien KD, Kuusisto J, Reichenbach DD, et al. Osteopontin is expressed in human aortic valvular lesions. Circulation 1995;92:2163–8. [3] Mohler ER, Chawla MK, Chang AW, et al. Identification and characterization of calcifying valve cells from human and canine aortic valves. J Heart Valve Dis 1999; 8:254–60. [4] Yetkin E, Waltenberger J. Molecular and cellular mechanisms of aortic stenosis. Int J Cardiol 2009;135(1):4–13. [5] Jian B, Narula N, Li Q, Mohler ERIII, Levy RJ. Progression of aortic valve stenosis: TGF-β1 is present in calcified aortic valve cusps and promotes aortic valve interstitial cell calcification via apoptosis. Ann Thorac Surg 2003;75(2):457–65. [6] Candeliere GA, Rao Y, Floh A, Sandler SD, Aubin JE. cDNA fingerprinting of osteoprogenitor cells to isolate differentiation stage-specific genes. Nucl Acids Res 1999;274:1079–83. [7] Shi GP, Sukhova GK, Grubb A, et al. Cystatin C deficiency in human atherosclerosis and aortic aneurysms. J Clin Invest 1999;104(9):1191–7. [8] Rajamannan NM, Subramaniam M, Rickard D, et al. Human aortic valve calcification is associated with an osteoblast phenotype. Circulation 2003;107(17):2181–4. [9] Waltenberger J, Lundin L, Öberg K, et al. Involvement of transforming growth factor-β in the formation of fibrotic lesions in carcinoid heart disease. Am J Pathol 1993;142:71–8.
[10] Kelm RJ, Hair GA, Mann KG, Grant BW. Characterization of human osteoblast and megakaryocyte-derived osteonectin (SPARC). Blood 1992;80:3112–9. [11] Johansson L, Grubb A, Abrahamson M, et al. A peptidyl derivative structurally based on the inhibitory center of cystatin C inhibits bone resorption in vitro. Bone 2000; 26(5):451–9. [12] Lerner UH, Grubb A. Human cystatin C, a cysteine proteinase inhibitor, inhibits bone resorption in vitro stimulated by parathyroid hormone and parathyroid hormonerelated peptide of malignancy. J Bone Miner Res 1992;7(4):433–40. [13] Lerner UH, Johansson L, Ranjso M, Rosenquist JB, Reinholt FP, Grubb A. Cystatin C, and inhibitor of bone resorption produced by osteoblasts. Acta Physiol Scand 1997;161(1):81–92. [14] Candeliere GA, Rao Y, Floh A, Sandler SD, Aubin JE. cDNA fingerprinting of osteoprogenitor cells to isolate differentiation stage-specific genes. Nucleic Acids Res 1999;27(4):1079–83. [15] Helske S, Syvaranta S, Lindstedt KA, et al. Increased expression of elastolytic cathepsins K, S, V and their inhibitor cystatin C in stenotic aortic valves. Arterioscler Thromb Vasc Biol 2006;26:1791–8. [16] Solem M, Rawson C, Lindburg K, Barnes D. Transforming growth factor beta regulates cystatin C in serum-free mouse embryo (SFME) cells. Biochem Biophys Res Commun 1990;172:945–51.
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
Increased expression of cystatin C and transforming growth factor β-1 in calcific aortic valves Ertan Yetkin a,⁎, Vadim Tchaikovski a, Nevzat Erdil b, Sadet Alan c, Johannes Waltenberger a a b c
Department of Cardiology, University Hospital Maastricht, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, Maastricht, The Netherlands Inonu University School of Medicine, Department of Cardiovascular Surgery, Malatya, Turkey Inonu University School of Medicine, Department of Pathology, Malatya, Turkey
a r t i c l e
i n f o
Article history: Received 25 June 2014 Accepted 27 July 2014 Available online 4 August 2014 Keywords: Cystatin C Growth factor Transforming growth factor Aortic valve Calcification aortic stenosis
Calcific aortic stenosis (AS) is a chronic and progressive disease. Until recently, the concept of calcific aortic valve disease as a degenerative and unmodifiable process basically induced by long-lasting mechanical stress was generally accepted [1] Studies have determined that mature lamellar bone formation occurs in calcified human aortic valves [2,3]. Yetkin and Waltenberger have reviewed potential molecular and cellular mechanisms of aortic stenosis in terms of extracellular matrix remodeling, angiogenesis, and osteoblast-like bone formation in aortic valve tissue [4]. Transforming growth factor beta-1 (TGF-β1) has been reported to play a role in the calcification of interstitial cells of aortic valves via promoting apoptosis [5]. TGF-β1 is a cytokine that has been demonstrated to promote calcification in human aortic valve interstitial cells [3] Cystatin-C is a cysteine protease inhibitor that was previously reported to be expressed in mature osteoblasts [6]. A potential link between Cys C and TGF-β1 has previously been reported in vascular smooth muscle cells [7]. Since the calcification process in human aortic valves resembles the mature lamellar bone formation, it is reasonable to expect the potential role of both molecules in the pathogenesis of calcification of human aortic valves. Accordingly, we aimed to evaluate the expression of TGF-β1 and Cys C in normal (non-calcified) and calcified human aortic valves. ⁎ Corresponding author at: Middle East Hospital, Mersin, Turkey.
http://dx.doi.org/10.1016/j.ijcard.2014.07.199 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.
Leaflets of tricuspid aortic valves were obtained from patients undergoing surgical aortic valve replacement because of calcific aortic stenosis (n = 6), from autopsy and organ donors (n = 4). Surgical specimens were obtained from 6 adults with an average age of 65 years. Aortic valve morphology was evaluated on hematoxylin and eosin stained sections. Cystatin C immunostaining: After deparaffinization process and blocking the endogenous peroxidase activity, sections were subjected to microwave treatment. Afterwards, sections were incubated for 90 min with polyclonal rabbit anti-human cystatin C antibody (1:1000, DAKO Cytomation, Glostrup; Denmark). After washing in TBS, biotinylated swine anti-rabbit IgG (1:150 Dako) was applied for 30 min as secondary antibody. After incubation with an alkaline phosphatase-coupled avidin– biotin complex (ABC complex, DAKO Cytomation, Glostrup; Denmark), both antibodies (TGF-β1 and cystatin C) were visualized with an alkaline substrate kit (Vector SK-5100, Vector Laboratories, Inc., UK). Parallel sections were stained with mouse monoclonal antibodies against TGF-β1 (1:500, Abcam). For the mouse monoclonal antibodies, biotinylated rabbit anti-mouse IgG (1:20) for 30 min was used as the secondary antibody, whereas biotinylated swine anti-rabbit IgG (1:200, Dako) was used as the secondary antibody for the polyclonal antibody. Sections were counterstained with hematoxylin and mounted with coverslips. In negative controls, incubation with primary antibody was omitted. Sections of immunostained specimens were evaluated by three independent observers using a semiquantitative scoring system: negative (−); focal, weak staining (+); present, strong staining (++); and very strong, widespread staining (+++). Western blot analysis of cystatin C and TGF-β1 expression: Crossslices of frozen aortic valves were homogenized on liquid nitrogen. After serial lysis and centrifugation process, supernatants containing 15 μg (cystatin C) or 100 μg (TGF-β1) of protein were heated in β-mercaptoethanol-containing sample buffer at 96 °C for 5 min and resolved on 20% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by a transfer onto nitrocellulose membranes (Amersham, Braunschweig, Germany). Subsequently, membranes were blocked with 5% semi-dry milk solution and incubated with antibody against cystatin C (DAKO Cytomation, Glostrup; Denmark) at a dilution of 1:3000 or TGF-β (Santa Cruz Biotechnology Inc., Santa Cruz,
E. Yetkin et al. / International Journal of Cardiology 176 (2014) 1252–1254
California; USA). The images were captured by scanning with LAS documentation system (Fuji Photo Film Co., Japan). Immunohistochemistry studies of six specimens of calcified aortic cusps revealed that both Cys C staining and TGF-β1 staining were strongly positive at calcified and non-calcified sites in both extracellular and cellular compartments, while those of normal aortic valves were largely negative for TGF-β1. Only the endothelial layer and fibrosa part of the normal aortic valve showed a mild staining for Cys C and TGF-β1. The pattern of Cys C and TGF-β1 immunostaining showed a close similarity, i.e., colocalization, regarding valvular structures such as endothelium and fibrosa, calcified and chondroid areas (Fig. 1), and capillarized and inflammatory areas. In normal human aortic valves, pathologic findings such as calcification, chondroid tissue, inflammatory area and capillary formation were not observed. Western blot analysis confirmed the increased expression of cystatin C in calcified aortic valve tissue compared to normal aortic valve tissue. In accordance with cystatin C expression, increased expression of TGF-β1 was detected in calcified aortic valve tissue compared to normal valve tissue. We have demonstrated that expression of TGF-β1 and cystatin C is significantly enhanced in calcified aortic human valves compared to normal human aortic valves. Additionally expression of TGF-β1 and cystatin C showed similar staining patterns in endothelium, extracellular matrix and inflammatory areas. Rajamannan et al. [8] have demonstrated that calcification in human aortic valve leaflets has similar features to that of osteoblastogenesis during skeletal bone formation. TGF-β1 is a cytokine that has been demonstrated to promote the calcification in human aortic valves [3]. TGF-β1 is characteristically present within calcified stenotic aortic cusps and mediates the calcification process of aortic valve interstitial cells in culture [5]. Similarly, the presence of TGF-β1 has also been shown in carcinoid plaques of the heart valves [9,10]. Cys C as a cysteine protease inhibitor was previously reported to be expressed in mature osteoblasts [6]. Additionally, Cys C has been found to inhibit bone resorption [11–13]. It has been shown that the maximum expression of Cys C appears to coincide with early
1253
events in osteoblast differentiation [14]. Recently, Helske et al. have demonstrated increased expression of elastolytic cathepsins, namely cathepsins K, S, and V and their inhibitor Cys C in stenotic human aortic valves [15]. Since we have demonstrated that expression of both TGF-β1 and Cys C is increased in calcified aortic valves, these molecules are likely to play a role in the calcification process of non-rheumatic aortic stenosis. It has been shown that TGF-β1 clearly regulates Cys C expression vascular smooth muscle [7]. A strong positive regulation of the Cys C gene by TGF-β1 in serum-free mouse embryonic cells has also been reported [16]. In conclusion, we have demonstrated that Cys C and TGF-β1 expression is significantly increased in non-rheumatic calcified aortic valves compared to normal aortic valve tissue. Both molecules have similar expression patterns. However, the mechanisms contributing to the interaction between Cys C and TGF-β1 in calcifying human aortic valve remain to be elucidated. Inhibiting the possible interaction between these two molecules could potentially enable us to prevent the progression of aortic valve calcification at an early stage of the disease.
Conflict of interest The authors report no relationships that could be construed as a conflict of interest.
Acknowledgment We would like to thank Anique Janssen for her technical and skilful assistance during the immunostaining procedure. The study was supported in part by an unrestricted grant from Medtronic BV. The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology.
Fig. 1. Similar expression pattern of Cys C and TGF-β1 in calcified aortic valve tissue. Immunohistochemical detection of Cys C in the calcifying (A1) and endothelial and subendothelial (A2) areas. Immunohistochemical detection of TGF-β1 in the calcifying (B1) and endothelial and subendothelial (B2) areas (primary magnification ×100).
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E. Yetkin et al. / International Journal of Cardiology 176 (2014) 1252–1254
References [1] Rosenhek R, Binder T, Porenta G, et al. Predictors of outcome in severe, asymptomatic aortic stenosis. N Engl J Med 2000;343:611–7. [2] O'Brien KD, Kuusisto J, Reichenbach DD, et al. Osteopontin is expressed in human aortic valvular lesions. Circulation 1995;92:2163–8. [3] Mohler ER, Chawla MK, Chang AW, et al. Identification and characterization of calcifying valve cells from human and canine aortic valves. J Heart Valve Dis 1999; 8:254–60. [4] Yetkin E, Waltenberger J. Molecular and cellular mechanisms of aortic stenosis. Int J Cardiol 2009;135(1):4–13. [5] Jian B, Narula N, Li Q, Mohler ERIII, Levy RJ. Progression of aortic valve stenosis: TGF-β1 is present in calcified aortic valve cusps and promotes aortic valve interstitial cell calcification via apoptosis. Ann Thorac Surg 2003;75(2):457–65. [6] Candeliere GA, Rao Y, Floh A, Sandler SD, Aubin JE. cDNA fingerprinting of osteoprogenitor cells to isolate differentiation stage-specific genes. Nucl Acids Res 1999;274:1079–83. [7] Shi GP, Sukhova GK, Grubb A, et al. Cystatin C deficiency in human atherosclerosis and aortic aneurysms. J Clin Invest 1999;104(9):1191–7. [8] Rajamannan NM, Subramaniam M, Rickard D, et al. Human aortic valve calcification is associated with an osteoblast phenotype. Circulation 2003;107(17):2181–4. [9] Waltenberger J, Lundin L, Öberg K, et al. Involvement of transforming growth factor-β in the formation of fibrotic lesions in carcinoid heart disease. Am J Pathol 1993;142:71–8.
[10] Kelm RJ, Hair GA, Mann KG, Grant BW. Characterization of human osteoblast and megakaryocyte-derived osteonectin (SPARC). Blood 1992;80:3112–9. [11] Johansson L, Grubb A, Abrahamson M, et al. A peptidyl derivative structurally based on the inhibitory center of cystatin C inhibits bone resorption in vitro. Bone 2000; 26(5):451–9. [12] Lerner UH, Grubb A. Human cystatin C, a cysteine proteinase inhibitor, inhibits bone resorption in vitro stimulated by parathyroid hormone and parathyroid hormonerelated peptide of malignancy. J Bone Miner Res 1992;7(4):433–40. [13] Lerner UH, Johansson L, Ranjso M, Rosenquist JB, Reinholt FP, Grubb A. Cystatin C, and inhibitor of bone resorption produced by osteoblasts. Acta Physiol Scand 1997;161(1):81–92. [14] Candeliere GA, Rao Y, Floh A, Sandler SD, Aubin JE. cDNA fingerprinting of osteoprogenitor cells to isolate differentiation stage-specific genes. Nucleic Acids Res 1999;27(4):1079–83. [15] Helske S, Syvaranta S, Lindstedt KA, et al. Increased expression of elastolytic cathepsins K, S, V and their inhibitor cystatin C in stenotic aortic valves. Arterioscler Thromb Vasc Biol 2006;26:1791–8. [16] Solem M, Rawson C, Lindburg K, Barnes D. Transforming growth factor beta regulates cystatin C in serum-free mouse embryo (SFME) cells. Biochem Biophys Res Commun 1990;172:945–51.
