ARTICLE |
Correspondence to: Alain-Pierre Gadeau, INSERM U441, Av du Haut Lévêque, 33600 Pessac, France. E-mail: alain.gadeau@bordeaux.inserm.fr
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Summary |
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Although mineral deposits have long been described to be a prominent feature of atherosclerosis, the mechanisms of arterial calcification are not well understood. However, accumulation of the non-collagenous matrix bone-associated proteins, osteopontin, osteocalcin, and osteonectin, has been demonstrated in atheromatous plaques. The aim of this study was to evaluate the role of these proteins in arterial calcification and, more precisely, during the initiation of this process. A model of rapid aortic calcification was developed in rabbits by an oversized balloon angioplasty. Calcification was followed using von Kossa staining and osteopontin, osteocalcin, and osteonectin were identified using immunohistochemistry. The aortic injury was rapidly followed by calcified deposits that appeared in the media as soon as 2 days after injury and then accumulated in zipper-like structures. Osteonectin was not detected in calcified deposits at any time after injury. In contrast, osteopontin and osteocalcin were detected in 8- and 14-day calcified structures, respectively, but not in the very early 2-day mineral deposits. These results suggest that these matrix proteins, osteopontin, osteocalcin, and osteonectin, are not involved in the initiation step of the aortic calcification process and that the former two might play a role in the regulation of arterial calcification. (J Histochem Cytochem 49:7986, 2001)
Key Words: atherosclerosis, calcifications, extracellular matrix, vessel wall
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Introduction |
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Arterial calcification is commonly considered to be a deleterious process involving passive calcium accumulation in the intima during the complication of atherosclerotic plaques or in the media during aging or in diabetic people. Studies of the ultrastructural and biochemical composition of calcium deposits in atherosclerotic plaques have demonstrated that they are mainly in the form of hydroxyapatite, which is the type of calcium crystal found in bone (
A large number of recent reports on the mechanisms of connective tissue mineralization and bone remodeling underline the potential role of three non-collagenous matrix proteins, osteopontin (OPN), osteonectin (ON), and osteocalcin (OC) (
OPN is an RGD-containing acidic phosphoprotein first isolated in bone (
OC is a vitamin K-dependent matrix protein that contains -carboxyglutamic acid. In vitro, this protein strongly inhibits calcium salt precipitation (
ON, also called SPARC or BM40, is a secreted calcium binding protein involved in bone development (
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Materials and Methods |
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Tissue Collection and Staining Method
Animal studies were performed in accordance with French ministry of agriculture guidelines. Aortic tissues were obtained from male Fauve de Bourgogne rabbits. Animals were divided into two groups. In the first group, 6 month-old rabbits (n = 7) were fed for 6 months with an 0.3% cholesterol-enriched diet and sacrificed. In the second group, 3 month-old rabbits (n = 15) were subjected to aortic balloon injury (1.21.5 atm, three times) with a 3F angioplasty balloon (Baxter; Irvine, CA) after anesthesia by IM administration of 20 mg/kg ketamine (Imalgene) (Merial; Lyon, France) and 2 mg/kg xylazin (Rompum) (Bayer Pharma; Puteaux, France). Three rabbits were sacrificed after a defined duration of recovery (2, 4, 8, 14, or 30 days). Aortas were quickly dissected from the descending aorta to the iliac branch and eight pieces (0.5 cm) were removed at the same level in each aorta. As a control, a piece of healthy aorta was removed in all rabbits, just under the aortic arch, and was checked for calcification. These controls were always free of calcified deposits. Moreover, a healthy rabbit aorta was harvested under the same conditions and used as control. Aortas were fixed in ice-cold 4% paraformaldehyde for at least 3 hr. Specimens were rinsed in PBS, then embedded in paraffin. Seven-µm-thick sections were spread over APES (3-aminopropyltriethoxy-silane; Sigma-Aldrich, SaintQuentin Fallavier, France)-coated slides.
Von Kossa-stained sections counterstained with Kernechtrot (Nuclear Fast Red) and Masson's trichrome were made according to classical methods (
Calcium deposits were also specifically stained with Alizarin Red S and the sections were examined with a confocal microscope (Nikon PCM 2000) as previously described (
Immunohistochemistry
Serial sections were used for immunocytochemistry. SMCs were detected using an SM -actin monoclonal antibody from Sigma-Aldrich, macrophages using RAM 11 monoclonal antibody from Dako (Trappes, France), OPN using MPIIIB10, and ON using AON-1 monoclonal antibodies purchased from DHSB, (Iowa City, IA) and OC using OCG2 from Takara (Biowhittaker Europe; Verviers, Belgium). Bound primary antibodies were detected using biotinylated anti-mouse secondary antibodies and the streptavidinbiotinylated horseradish peroxidase complex (Amersham Pharmacia Biotech; Orsay, France). The final complex was visualized by treatment with PBS containing 0.5 mg/ml diaminobenzidine and 0.03% H2O2 (Sigma-Aldrich). Antibody dilution and washes were carried out in PBS buffer containing 0.2% Tween-20 (Sigma-Aldrich) and 0.5% bovine serum albumin (ICN Biomedicals; Orsay, France). Controls were performed without primary antibodies and with nonspecific IgG1 antibody. To verify that antibodies did not bind nonspecifically to the calcium deposits, control sections with large calcifications were decalcified by 3% citric acid (Prolabo; Bordeaux, France) treatment for 1 hr before immunolabeling (
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Results |
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OPN, OC, and ON Accumulation in Rabbit Atherosclerotic Plaques
Aortic calcification of cholesterol-enriched diet-fed rabbit occurred frequently (
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Because hydroxyapatite has a high binding capacity for many proteins, we verified whether OPN and OC immunodetection found in calcified deposits was really due to the presence of OPN and OC antigens. Calcium phosphate was removed by a 1-hr citric acid treatment. Completion of calcium removal was controlled by the absence of black stain deposit after von Kossa staining (Fig 1D). However, a residual material, detected by Kernechtrot counterstaining (a nuclear red and cytoplasmic pink stain) remained in place of the calcium deposit. After this treatment, OC and OPN were still detected in the same areas with a nearly equal staining intensity to that in untreated sections (Fig 1E vs 1B and 1F vs 1C), demonstrating that the antibodies were not nonspecifically adsorbed on hydroxyapatite.
When primary antibody was omitted or replaced by nonspecific IgG1, the second antibody or streptavidinperoxidase complex did not bind to calcifications (not shown).
Early Events in the Mineral Deposits
Early events in the mineral deposits were monitored in the rabbit aorta balloon injury model. The aortic injury was performed at 1.21.5 atm by moving a 3F angioplasty balloon from the thoracic to the abdominal aorta. Aortas were harvested either before injury or 2, 4, 8, 14, or 30 days after injury, and serial sections were made. Immunostaining with SM -actin and RAM 11 antibodies demonstrated that only SMCs were detected in the injured tissue. At Day 30, some macrophages were observed in association with the calcification (not shown).
Masson's trichrome staining showed extensive structural tissue reorganization of the media in comparison to healthy aorta (Fig 2A). Two days and 4 days after injury, the interlaminae spaces mainly located in the inner half of the media were devoid of cells, as demonstrated by the absence of red cell staining (Fig 2B and Fig 2C). Sections of 8, 14, and 30 days were torn, suggesting the presence of "hard" components, such as calcified materials, in the media (Fig 2D shows a 14-day picture). Indeed, von Kossa's staining of serial sections showed that the arterial wound induced medial calcification. Although calcifications were never observed in healthy tissue (Fig 2E), 2 days after injury calcium deposits were found in the aorta of two of the three rabbits (Fig 2F). These deposits were not homogeneously distributed in the media but appeared only in cell-free areas. High magnification showed that calcified deposits were first present between the elastic laminae, mainly in association with remaining cellular materials stained by Kernechtrot (not shown). Confocal microscopic examination after Alizarin Red S treatment confirmed that the first calcified deposits (red fluorescence) were not closely associated with the elastic laminae (green autofluorescence) (Fig 2I). On Day 4, more extended calcifications were also detected in the media of two rabbits (Fig 2G), and deposits began to fill the empty areas in place of the cells between the elastic laminae. From Day 8, the elastic laminae were totally embedded in the calcified structure (not shown) and became fragmented into small pieces, and large calcium deposits demonstrating a dense, zipper-like morphology were present on Day 14 (Fig 2H). Therefore, calcium deposits rapidly increased between Days 2 and 4 after injury from 2.520% of the total media surface, reaching more than 30% 1 month later (Fig 3).
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Successive sections of injured aortas were investigated for OPN, OC, and ON protein location. OPN was not detected in healthy media (Fig 4A) but it appeared very rapidly 2 days after injury (Fig 4B). OPN was detected only in the remaining cells of injured media. In the same way, 4 days after injury, OPN was essentially detected in SMCs of the middle and internal part of the media (Fig 4C). At that time, OPN accumulation was not associated with very early calcified deposits. Then, from Day 8, OPN was detected in the neointima and also in the media, where it was clearly associated with calcified areas (Fig 4D).
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In healthy rabbit and before the eighth day after injury, OC was not detected in the media (Fig 5A5C). Then, from Day 14 after injury, OC accumulated in association with calcium deposits (Fig 5D) and began to be detected in SMCs. In healthy tissue, ON was detected only in endothelial cells and adventitia (Fig 6A), but 14 days after injury this protein was found in the intima, in the cellular part of the media, and all around but not inside calcifications (Fig 6B).
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Discussion |
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To understand the role of OPN, OC, and ON accumulation in calcified areas and their involvement in the initiation of this process, we developed a model of rapid aortic calcification that enabled us to follow non-collagenous bone proteins throughout the development of aortic calcification. In this model, severe aortic injury was induced by balloon angioplasty.
Surprisingly, calcified deposits appeared very rapidly, only 24 days after injury, in areas of the injured aortic media where cells had been killed. This rapid phenomenon, detected only in dead-cell areas, suggests that the early initiation of calcification is linked to SMC death, unveiling necrotic areas which can function as nidus for this process. During balloon injury a large number of cells were killed by crushing and by apoptosis (
Our results suggest that the early steps of the calcification process after rabbit injury could be related to cell death. In this case, dead cells might create an active nucleation of calcium crystals. The mechanisms involved in the subsequent steps are not clearly understood but could involve the bone-associated matrix proteins detected in calcified deposits, OPN, OC, matrix Gla protein (MGP) (
Two weeks after injury, all calcified deposits contained OPN and OC, suggesting that these proteins were adsorbed on the calcium crystals because of their strong affinity for hydroxyapatite. This observation indicates that these proteins could be involved in the regulation of calcium crystal development. Although
OPN and OC could come from the surrounding cells or from plasma that has infiltrated the injured aortic media. In the calcification of human atherosclerotic plaques, it has been shown that OPN may be of macrophage or SMC origin (
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Acknowledgments |
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Supported by grants from Institut National de la Santé et de la Recherche Médicale.
We wish to thank P. Alzieu for excellent technical assistance. Monoclonal antibodies MPIIIB 10 (1) developed by M. Solursh/A. Franzen and AON-1 developed by J.D. Termine were obtained from the Developmental Studies Hybridoma Bank under the auspices of NICHD and maintained by the University of Iowa Department of Biological Sciences (Iowa City, IA).
Received for publication March 28, 2000; accepted August 23, 2000.
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