Ultrastructural Pathology, 2014; 38(4): 281–284 ! Informa Healthcare USA, Inc. ISSN: 0191-3123 print / 1521-0758 online DOI: 10.3109/01913123.2014.901468
ORIGINAL ARTICLE
Collagen Mineralization in Human Aortic Valve Stenosis: A Field Emission Scanning Electron Microscopy and Energy Dispersive Spectroscopy Analysis Ida Perrotta, PhD and Mariano Davoli Department of Biology, Ecology and Earth Science (Di.B.E.S.T.), Laboratory of Electron Microscopy (cubo 15A), University of Calabria, Cosenza, Italy
ABSTRACT Calcific aortic stenosis is a slowly progressive disorder characterized by an important extracellular matrix remodeling with fibrosis and massive deposition of minerals (primarily calcium) in the valve leaflet. The main structural components of human aortic valve are the large, thick collagen bundles that withstand the diastolic loading. Collagen has been studied in a number of reports that aim to clarify the mechanisms underlying the structural deterioration of heart valve substitutes, however to date, little is known regarding the morphological interaction between collagen and mineral crystals in the calcifying tissue of native aortic valve. Here, we have analyzed a total of 12 calcified native aortic valves by using scanning electron microscopy (SEM) with Energy Dispersive X-Ray Analysis (EDX) to depict the morphological appearance of mineralized collagen and to determine the location of calcium phosphate minerals in the collagen matrix of the valve cusp. Our results demonstrate that crystals probably nucleate and grow in the interior of the collagen fibers in the absence of surface events. Keywords: Calcific aortic stenosis, energy-dispersive X-ray spectroscopy, scanning electron microscopy
and secondary (SE) electron imaging, provides general information on the gross morphology of the valve leaflet allowing an easy discrimination between the different organic and inorganic phases present in the samples analyzed. This method also makes it possible to show details and/or structures down to the collagenous matrix or inside the collagen fibers themselves that have so far only been routinely visualized by sectioning the specimen.
Calcific aortic stenosis (CAS) is the most common form of valve disease in the Western world, strongly related to aging, and histologically characterized by diffuse fibrous thickening, distortion of the valve leaflet and extensive calcification [1]. Although first described by Monckeberg nearly a century ago, to date little is known regarding the molecular and cellular events that control the stenosing process with only a few reports dealing with the structural appearance of mineralized collagen in the calcifying tissue of native aortic valve [2–4]. Therefore, in the present study we have investigated the morphological association between collagen and minerals in human aortic valves that undergo dystrophic calcification by using scanning electron microscopy (SEM) with energy dispersive X-ray analysis (EDX). Our approach, based on the use of backscattered (BSE)
STUDY MATERIALS Calcified native aortic valves were collected from 12 patients (5 male and 7 female; mean age, 55.1 ± 4.7 years) who underwent aortic valve replacement for CAS at the Sant’Anna Hospital (Department of
Received 23 February 2014; Accepted 3 March 2014; Published online 15 May 2014 Correspondence: Dr. Ida Perrotta, Department of Biology, Ecology and Earth Science, Laboratory of Electron Microscopy, University of Calabria, Arcavacata di Rende, Cosenza 87036, Italy. Tel: +39(0)984 493632. E-mail: [email protected]
281
282 I. Perrotta and M. Davoli Cardiology and Heart Surgery, Catanzaro, Italy). Patients with rheumatic or congenital valve disease were excluded from the study design. Clinical records were reviewed for cardiovascular risk factors, hyperparathyroidism and previous history of peripheral arterial disease.
METHODS With institutional review board approval and patient consent, calcified valve tissues were fixed in 3% glutaraldehyde, post-fixed in OsO4 and dehydrated in graded ethanol solutions. Samples were subsequently transferred to 100% hexamethyldisilazane (HMDS) through a graded series of ethanol–HMDS mixtures, air-dried at room temperature and mounted on aluminium stubs with double sticky tabs. Tissues were coated with 10 nm graphite, and examined with a FEI Quanta 200 ESEM. Imaging was performed in high vacuum at 20 kV.
RESULTS In the calcified valves analyzed in our series, collagen fibers exhibited an irregular profile with a multiplicity of excrescences or protuberances on their external surface (arrowheads and asterisk in Figure 1A). Elemental analysis (Figure 1B) revealed that these abnormal formations principally consisted of calcium and phosphate, thus resembling the chemical composition of hydroxyapatite. By comparing SEM SE
(Figure 2A–C) and BSE (Figure 2D–F) images, these calcium deposits appeared as bright, regular spheres located within the collagen fibers and protruding from their surface as spiculated masses with microlobulated or frankly margins. Examination of heavily calcified areas revealed the presence of numerous dense structures of irregular shapes made up of single, spheroidal particles of hydroxyapatite arranged in grape-like clusters and embedded in the collagen matrix of the cusp (Figure 3). There was no evidence of mineral deposition onto collagen fibers surface and/or between them in the extracellular space.
DISCUSSION CAS is the most common form of valve disease and, currently, the main indication for aortic valve replacement in developed countries. Although initially considered an age-associated degenerative phenomenon during which serum calcium passively attaches to the surface of the valve cusp, recent data have established that CAS is a regulated form of tissue mineralization involving extracellular matrix (ECM) alterations and mechanisms of bone formation within the leaflet [5,6]. Aortic valves are composed primarily of collagen fibers, representing 50% of the total ECM. Collagen remodeling has been well-studied in atherosclerosis and although it is becoming increasingly clear that it might also play important roles in the pathogenesis of aortic valve stenosis, to date relatively little attention has been directed to certain
FIGURE 1. Within the calcified valve leaflet, collagen shows an irregular profile with gross protuberances and knobby excrescences (asterisk and arrowheads) (A, image formed from secondary electrons). EDX analysis demonstrates that these abnormal formations principally contain calcium and phosphorus with trace amounts of magnesium (Mg), sodium (Na) and sulfur (S) (B). Ultrastructural Pathology
Collagen calcification in CAS 283
FIGURE 2. Secondary electrons images showing the morphological appearance of mineralized collagen in the diseased valves (A–C); bottom panel (D–F) displays the corresponding images recorded with BSE and demonstrating the presence of round-shaped mineral deposits within the collagen fibers of the valve. Note that the knobby excrescences seen in Figure 1(A–C) on the surface of the collagen correspond to the bright mineral spheres in the BSE images (Figure 1D–F). Ca-P crystals seem to be entrapped in the collagen fiber network of the leaflet; no identifiable crystal features are seen on the surface of these fibers.
FIGURE 3. Secondary (A) and back-scattered (B) electron images showing crystals of hydroxyapatite forming large and irregular, grape-like clusters embedded in the collagen matrix of the valve cusp.
microstructural characteristics of collagen fibers in the calcified heart valves [2–4,7]. In this context, the pathobiology of collagen has been mainly investigated in the tissue engineering research area, with the goal !
2014 Informa Healthcare USA, Inc.
to identify and characterize the mechanisms governing the structural deterioration of prosthetic and bioprosthetic heart valves substitutes [8,9]. For instance, it has been shown that collagen is able to
284 I. Perrotta and M. Davoli undergo a time-dependent process of focal calcification and that calcified nodules are composed of hydroxyapatite crystals precipitated on a collagenous matrix. Here, we documented for the first time to the best of our knowledge the morphological interaction between collagen and crystals as well as the location of these mineral particles in the calcified tissues of native aortic valves. Our analyses demonstrate that within the diseased heart valve cusps, mineralization of the collagen-matrix may probably begin within the collagen fibers trought a mechanism similar to that previously described for bone tissue [10]. This hypothesis is supported by our electron microscopic results demonstrating the presence of small roundship aggregates of hydroxyapatite within the collagen fibers with no evidence of Ca-P crystals upon the fibers and/or between them in the extracellular space. In addition, the presence of multiple, small mineral deposits within individual collagen fibers seem to suggest that crystal formation might occur in a temporal and spatial independent manner. From a chemical point of view, as expected, valve minerals mainly consist of hydroxyapatite but contain magnesium, sodium and sulfur impurities. Although our data have to be taken as mainly descriptive and preliminary, the presence of collagen fibers having crystals principally in their interior seems to suggest that minerals may nucleate and grow within the collagen fibers themselves, in the absence of surface events. Such results may provide new insights into the possible mechanism of collagen-mediated mineralization in the valve leaflet and, in general, in the calcifying tissues and may serve as a base for future studies, as this process has important effects on the development and progression of CAS.
ACKNOWLEDGEMENTS The authors thank Dr Alfonso Sciangula (Sant’Anna Hospital, Catanzaro, Italy) who kindly provided clinical specimens.
DECLARATION OF INTEREST Authors certify that there is no conflict of interest that would prejudice the impartiality of this scientific work. No specific funding has been received for this work.
REFERENCES 1. Perrotta I, Russo E, Camastra C, et al. New evidence for a critical role of elastin in calcification of native heart valves: Immunohistochemical and ultrastructural study with literature review. Histopathology 2011;59: 504–513. 2. Monckeberg JG. Der normale histologische Bau und die Sklerose der Aortenklappen. Virchows Arch Pathol Anat Physiol 1904;176: 472–514. 3. Delogne C, Lawford PV, Habesch SM, Carolan VA. Characterization of the calcification of cardiac valve bioprostheses by environmental scanning electron microscopy and vibrational spectroscopy. J Microsc 2007;228: 62–77. 4. Weska RF, Aimoli CG, Nogueira GM, et al. Natural and prosthetic heart valve calcification: Morphology and chemical composition characterization. Artif Organs 2010; 34: 311–318. 5. Chen JH, Simmons CA. Cell–matrix interactions in the pathobiology of calcific aortic valve disease: Critical roles for matricellular, matricrine, and matrix mechanics cues. Circ Res 2011;108: 1510–1524. 6. Towler DA. Vascular calcification: A perspective on an imminent disease epidemic. IBMS BoneKEy 2008;5: 41–58. 7. Adiguzel E, Ahmad PJ, Franco C, Bendeck MP. Collagens in the progression and complications of atherosclerosis. Vasc Med 2009;14: 73–89. 8. Vyavahare N, Hirsch D, Lerner E, et al. Prevention of bioprosthetic heart valve calcification by ethanol preincubation. Efficacy and mechanisms. Circulation 1997;95: 479–488. 9. Waller BF, McKay C, Van Tassel J, Allen M. Catheter balloon valvuloplasty of stenotic porcine bioprosthetic valves: Part I: Anatomic considerations. Clin Cardiol 1991; 14: 686–691. 10. Price PA, Toroian D, Lim JE. Mineralization by inhibitor exclusion: The calcification of collagen with fetuin. J Biol Chem 2009;284: 17092–17101.
Ultrastructural Pathology
Copyright of Ultrastructural Pathology is the property of Taylor & Francis Ltd and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
ORIGINAL ARTICLE
Collagen Mineralization in Human Aortic Valve Stenosis: A Field Emission Scanning Electron Microscopy and Energy Dispersive Spectroscopy Analysis Ida Perrotta, PhD and Mariano Davoli Department of Biology, Ecology and Earth Science (Di.B.E.S.T.), Laboratory of Electron Microscopy (cubo 15A), University of Calabria, Cosenza, Italy
ABSTRACT Calcific aortic stenosis is a slowly progressive disorder characterized by an important extracellular matrix remodeling with fibrosis and massive deposition of minerals (primarily calcium) in the valve leaflet. The main structural components of human aortic valve are the large, thick collagen bundles that withstand the diastolic loading. Collagen has been studied in a number of reports that aim to clarify the mechanisms underlying the structural deterioration of heart valve substitutes, however to date, little is known regarding the morphological interaction between collagen and mineral crystals in the calcifying tissue of native aortic valve. Here, we have analyzed a total of 12 calcified native aortic valves by using scanning electron microscopy (SEM) with Energy Dispersive X-Ray Analysis (EDX) to depict the morphological appearance of mineralized collagen and to determine the location of calcium phosphate minerals in the collagen matrix of the valve cusp. Our results demonstrate that crystals probably nucleate and grow in the interior of the collagen fibers in the absence of surface events. Keywords: Calcific aortic stenosis, energy-dispersive X-ray spectroscopy, scanning electron microscopy
and secondary (SE) electron imaging, provides general information on the gross morphology of the valve leaflet allowing an easy discrimination between the different organic and inorganic phases present in the samples analyzed. This method also makes it possible to show details and/or structures down to the collagenous matrix or inside the collagen fibers themselves that have so far only been routinely visualized by sectioning the specimen.
Calcific aortic stenosis (CAS) is the most common form of valve disease in the Western world, strongly related to aging, and histologically characterized by diffuse fibrous thickening, distortion of the valve leaflet and extensive calcification [1]. Although first described by Monckeberg nearly a century ago, to date little is known regarding the molecular and cellular events that control the stenosing process with only a few reports dealing with the structural appearance of mineralized collagen in the calcifying tissue of native aortic valve [2–4]. Therefore, in the present study we have investigated the morphological association between collagen and minerals in human aortic valves that undergo dystrophic calcification by using scanning electron microscopy (SEM) with energy dispersive X-ray analysis (EDX). Our approach, based on the use of backscattered (BSE)
STUDY MATERIALS Calcified native aortic valves were collected from 12 patients (5 male and 7 female; mean age, 55.1 ± 4.7 years) who underwent aortic valve replacement for CAS at the Sant’Anna Hospital (Department of
Received 23 February 2014; Accepted 3 March 2014; Published online 15 May 2014 Correspondence: Dr. Ida Perrotta, Department of Biology, Ecology and Earth Science, Laboratory of Electron Microscopy, University of Calabria, Arcavacata di Rende, Cosenza 87036, Italy. Tel: +39(0)984 493632. E-mail: [email protected]
281
282 I. Perrotta and M. Davoli Cardiology and Heart Surgery, Catanzaro, Italy). Patients with rheumatic or congenital valve disease were excluded from the study design. Clinical records were reviewed for cardiovascular risk factors, hyperparathyroidism and previous history of peripheral arterial disease.
METHODS With institutional review board approval and patient consent, calcified valve tissues were fixed in 3% glutaraldehyde, post-fixed in OsO4 and dehydrated in graded ethanol solutions. Samples were subsequently transferred to 100% hexamethyldisilazane (HMDS) through a graded series of ethanol–HMDS mixtures, air-dried at room temperature and mounted on aluminium stubs with double sticky tabs. Tissues were coated with 10 nm graphite, and examined with a FEI Quanta 200 ESEM. Imaging was performed in high vacuum at 20 kV.
RESULTS In the calcified valves analyzed in our series, collagen fibers exhibited an irregular profile with a multiplicity of excrescences or protuberances on their external surface (arrowheads and asterisk in Figure 1A). Elemental analysis (Figure 1B) revealed that these abnormal formations principally consisted of calcium and phosphate, thus resembling the chemical composition of hydroxyapatite. By comparing SEM SE
(Figure 2A–C) and BSE (Figure 2D–F) images, these calcium deposits appeared as bright, regular spheres located within the collagen fibers and protruding from their surface as spiculated masses with microlobulated or frankly margins. Examination of heavily calcified areas revealed the presence of numerous dense structures of irregular shapes made up of single, spheroidal particles of hydroxyapatite arranged in grape-like clusters and embedded in the collagen matrix of the cusp (Figure 3). There was no evidence of mineral deposition onto collagen fibers surface and/or between them in the extracellular space.
DISCUSSION CAS is the most common form of valve disease and, currently, the main indication for aortic valve replacement in developed countries. Although initially considered an age-associated degenerative phenomenon during which serum calcium passively attaches to the surface of the valve cusp, recent data have established that CAS is a regulated form of tissue mineralization involving extracellular matrix (ECM) alterations and mechanisms of bone formation within the leaflet [5,6]. Aortic valves are composed primarily of collagen fibers, representing 50% of the total ECM. Collagen remodeling has been well-studied in atherosclerosis and although it is becoming increasingly clear that it might also play important roles in the pathogenesis of aortic valve stenosis, to date relatively little attention has been directed to certain
FIGURE 1. Within the calcified valve leaflet, collagen shows an irregular profile with gross protuberances and knobby excrescences (asterisk and arrowheads) (A, image formed from secondary electrons). EDX analysis demonstrates that these abnormal formations principally contain calcium and phosphorus with trace amounts of magnesium (Mg), sodium (Na) and sulfur (S) (B). Ultrastructural Pathology
Collagen calcification in CAS 283
FIGURE 2. Secondary electrons images showing the morphological appearance of mineralized collagen in the diseased valves (A–C); bottom panel (D–F) displays the corresponding images recorded with BSE and demonstrating the presence of round-shaped mineral deposits within the collagen fibers of the valve. Note that the knobby excrescences seen in Figure 1(A–C) on the surface of the collagen correspond to the bright mineral spheres in the BSE images (Figure 1D–F). Ca-P crystals seem to be entrapped in the collagen fiber network of the leaflet; no identifiable crystal features are seen on the surface of these fibers.
FIGURE 3. Secondary (A) and back-scattered (B) electron images showing crystals of hydroxyapatite forming large and irregular, grape-like clusters embedded in the collagen matrix of the valve cusp.
microstructural characteristics of collagen fibers in the calcified heart valves [2–4,7]. In this context, the pathobiology of collagen has been mainly investigated in the tissue engineering research area, with the goal !
2014 Informa Healthcare USA, Inc.
to identify and characterize the mechanisms governing the structural deterioration of prosthetic and bioprosthetic heart valves substitutes [8,9]. For instance, it has been shown that collagen is able to
284 I. Perrotta and M. Davoli undergo a time-dependent process of focal calcification and that calcified nodules are composed of hydroxyapatite crystals precipitated on a collagenous matrix. Here, we documented for the first time to the best of our knowledge the morphological interaction between collagen and crystals as well as the location of these mineral particles in the calcified tissues of native aortic valves. Our analyses demonstrate that within the diseased heart valve cusps, mineralization of the collagen-matrix may probably begin within the collagen fibers trought a mechanism similar to that previously described for bone tissue [10]. This hypothesis is supported by our electron microscopic results demonstrating the presence of small roundship aggregates of hydroxyapatite within the collagen fibers with no evidence of Ca-P crystals upon the fibers and/or between them in the extracellular space. In addition, the presence of multiple, small mineral deposits within individual collagen fibers seem to suggest that crystal formation might occur in a temporal and spatial independent manner. From a chemical point of view, as expected, valve minerals mainly consist of hydroxyapatite but contain magnesium, sodium and sulfur impurities. Although our data have to be taken as mainly descriptive and preliminary, the presence of collagen fibers having crystals principally in their interior seems to suggest that minerals may nucleate and grow within the collagen fibers themselves, in the absence of surface events. Such results may provide new insights into the possible mechanism of collagen-mediated mineralization in the valve leaflet and, in general, in the calcifying tissues and may serve as a base for future studies, as this process has important effects on the development and progression of CAS.
ACKNOWLEDGEMENTS The authors thank Dr Alfonso Sciangula (Sant’Anna Hospital, Catanzaro, Italy) who kindly provided clinical specimens.
DECLARATION OF INTEREST Authors certify that there is no conflict of interest that would prejudice the impartiality of this scientific work. No specific funding has been received for this work.
REFERENCES 1. Perrotta I, Russo E, Camastra C, et al. New evidence for a critical role of elastin in calcification of native heart valves: Immunohistochemical and ultrastructural study with literature review. Histopathology 2011;59: 504–513. 2. Monckeberg JG. Der normale histologische Bau und die Sklerose der Aortenklappen. Virchows Arch Pathol Anat Physiol 1904;176: 472–514. 3. Delogne C, Lawford PV, Habesch SM, Carolan VA. Characterization of the calcification of cardiac valve bioprostheses by environmental scanning electron microscopy and vibrational spectroscopy. J Microsc 2007;228: 62–77. 4. Weska RF, Aimoli CG, Nogueira GM, et al. Natural and prosthetic heart valve calcification: Morphology and chemical composition characterization. Artif Organs 2010; 34: 311–318. 5. Chen JH, Simmons CA. Cell–matrix interactions in the pathobiology of calcific aortic valve disease: Critical roles for matricellular, matricrine, and matrix mechanics cues. Circ Res 2011;108: 1510–1524. 6. Towler DA. Vascular calcification: A perspective on an imminent disease epidemic. IBMS BoneKEy 2008;5: 41–58. 7. Adiguzel E, Ahmad PJ, Franco C, Bendeck MP. Collagens in the progression and complications of atherosclerosis. Vasc Med 2009;14: 73–89. 8. Vyavahare N, Hirsch D, Lerner E, et al. Prevention of bioprosthetic heart valve calcification by ethanol preincubation. Efficacy and mechanisms. Circulation 1997;95: 479–488. 9. Waller BF, McKay C, Van Tassel J, Allen M. Catheter balloon valvuloplasty of stenotic porcine bioprosthetic valves: Part I: Anatomic considerations. Clin Cardiol 1991; 14: 686–691. 10. Price PA, Toroian D, Lim JE. Mineralization by inhibitor exclusion: The calcification of collagen with fetuin. J Biol Chem 2009;284: 17092–17101.
Ultrastructural Pathology
Copyright of Ultrastructural Pathology is the property of Taylor & Francis Ltd and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
