Has our understanding of calcification in human coronary atherosclerosis progressed?

Abstract

Coronary artery calcification is a well-established predictor of future cardiac events; however, it is not a predictor of unstable plaque. The intimal calcification of the atherosclerotic plaques may begin with smooth muscle cell apoptosis and release of matrix vesicles and is almost always seen microscopically in pathological intimal thickening, which appears as microcalcification (≥0.5 μm, typically <15 μm in diameter). Calcification increases with macrophage infiltration into the lipid pool in early fibroatheroma where they undergo apoptosis and release matrix vesicles. The confluence of calcified areas involves extracellular matrix and the necrotic core, which can be identified by radiography as speckled (≤2 mm) or fragmented (>2, <5 mm) calcification. The calcification in thin-cap fibroatheromas and plaque rupture is generally less than what is observed in stable plaques and is usually speckled or fragmented. Fragmented calcification spreads into the surrounding collagen-rich matrix forming calcified sheets, the hallmarks of fibrocalcific plaques. The calcified sheets may break into nodules with fibrin deposition, and when accompanied by luminal protrusion, it is associated with thrombosis. Calcification is highest in fibrocalcific plaques followed by healed plaque rupture and is the least in erosion and pathological intimal thickening. The extent of calcification is greater in men than in women especially in the premenopausal period and is also greater in whites compared with blacks. The mechanisms of intimal calcification remain poorly understood in humans. Calcification often occurs in the presence of apoptosis of smooth muscle cells and macrophages with matrix vesicles accompanied by expression of osteogenic markers within the vessel wall.

Keywords: coronary artery disease; pathology; vascular calcification.

Figure 1

Progression of coronary calcification. Non-decalcified arterial segments (A and B) and decalcified segments (C to J) were serially cut for the microscopic assessment. A shows pathologic intimal thickening (PIT) characterized by lipid pool (LP) that lacks smooth muscle cells (SMCs) (negative for α-smooth muscle actin [α-SMA]) and shows the presence of apoptotic SMCs which can be identified by prominent basement membrane which stains positive with periodic acid Schiff (PAS) and the arrows point to in the high-power image (top right corner). Early microcalcification (≥0.5 μm, typically <15 μm in diameter) likely results from SMC apoptosis and calcification is detected by von Kossa staining within the LP (corresponding with a boxed area in the Movat image) where bone related proteins such as osteoprotegerin (OPG), osteopontin (OPN), and matrix Gla protein (MGP) are detected. Early necrotic core (NC) (B) not only lacks SMCs but is infiltrated by macrophages which eventually undergo apoptosis and calcification, which is observed as punctate (≥15 μm) areas of calcification. The microcalcification in early NC show variable amounts of staining for macrophage CD68 antigen; however, von Kossa staining clearly shows relatively larger punctate areas of calcification resulting from macrophage cell death within the NC as compared to microcalcification of dying SMCs. These calcified macrophages show co-localization of bone related proteins. Substantial amount of macrophage calcification can be observed in early NC (C) but the degree of calcification in NC typically increases towards the medial wall where fragmented calcifications can be seen (D). Microcalcification resulting from macrophage or SMC deaths can also be detected within a thin-fibrous cap and may be associated with plaque rupture (E). Calcification generally progress into the surrounding area of the NC (F), which leads to the development of sheets of calcification where both collagen matrix (G) and necrotic core itself are calcified (H). Nodular calcification may occur within the plaque in the absence of luminal thrombus and is characterized by breaks in calcified plates with fragments of calcium separated by fibrin (I). Ossification may occur at the edge of an area of calcification especially in nodular calcification (J). Immunohistochemical stainings in A and B were performed with the use of antibodies for CD68 (dilution 1:800; Dako, Carpinteria, CA), α-SMA (dilution 1:400, Dako), OPG (dilution 1:50, Novus Biologicals, LLC, Littleton, CO), OPN (dilution 1:400, generously provided by Larry W. Fisher, PhD, National Institute of Health, Bethesda, MD), and MGP (dilution 1:200, Enzo Life Science, Farmingdale, NY), respectively.

Figure 1

Progression of coronary calcification. Non-decalcified arterial segments (A and B) and decalcified segments (C to J) were serially cut for the microscopic assessment. A shows pathologic intimal thickening (PIT) characterized by lipid pool (LP) that lacks smooth muscle cells (SMCs) (negative for α-smooth muscle actin [α-SMA]) and shows the presence of apoptotic SMCs which can be identified by prominent basement membrane which stains positive with periodic acid Schiff (PAS) and the arrows point to in the high-power image (top right corner). Early microcalcification (≥0.5 μm, typically <15 μm in diameter) likely results from SMC apoptosis and calcification is detected by von Kossa staining within the LP (corresponding with a boxed area in the Movat image) where bone related proteins such as osteoprotegerin (OPG), osteopontin (OPN), and matrix Gla protein (MGP) are detected. Early necrotic core (NC) (B) not only lacks SMCs but is infiltrated by macrophages which eventually undergo apoptosis and calcification, which is observed as punctate (≥15 μm) areas of calcification. The microcalcification in early NC show variable amounts of staining for macrophage CD68 antigen; however, von Kossa staining clearly shows relatively larger punctate areas of calcification resulting from macrophage cell death within the NC as compared to microcalcification of dying SMCs. These calcified macrophages show co-localization of bone related proteins. Substantial amount of macrophage calcification can be observed in early NC (C) but the degree of calcification in NC typically increases towards the medial wall where fragmented calcifications can be seen (D). Microcalcification resulting from macrophage or SMC deaths can also be detected within a thin-fibrous cap and may be associated with plaque rupture (E). Calcification generally progress into the surrounding area of the NC (F), which leads to the development of sheets of calcification where both collagen matrix (G) and necrotic core itself are calcified (H). Nodular calcification may occur within the plaque in the absence of luminal thrombus and is characterized by breaks in calcified plates with fragments of calcium separated by fibrin (I). Ossification may occur at the edge of an area of calcification especially in nodular calcification (J). Immunohistochemical stainings in A and B were performed with the use of antibodies for CD68 (dilution 1:800; Dako, Carpinteria, CA), α-SMA (dilution 1:400, Dako), OPG (dilution 1:50, Novus Biologicals, LLC, Littleton, CO), OPN (dilution 1:400, generously provided by Larry W. Fisher, PhD, National Institute of Health, Bethesda, MD), and MGP (dilution 1:200, Enzo Life Science, Farmingdale, NY), respectively.

Figure 2

Advanced human atherosclerotic plaque showing apoptotic smooth muscle cells (SMCs). A is stained by the TUNEL technique and B is the same TUNEL-labeled section subsequently stained by PAS (periodic acid-Schiff) stain (TUNEL+PAS technique). A labeled nucleus is present in a cell–poor area in the fibrous cap (A), and this TUNEL-positive nucleus belongs to a cell that is surrounded by a prominent cage of PAS-positive basal laminae (B). This points to an SMC undergoing apoptotic cell death. Most of the SMCs in this region do not express α-SMC actin. Adjacent to this cell are PAS-positive empty cages of thickened basal lamina. C is transmission electron microscopy (TEM) image of an advanced human atherosclerotic plaque. Two SMCs are demonstrated that are completely disintegrated into myriad vesicles (granulovesicular degeneration). The prominent basal laminae (bl) around these clusters of vesicles led us to conclude that the vesicles are of SMC and not of macrophage origin. (Reproduced with permission from Kockx MM, et al. Circulation. 1998;97:2307–2315.)

Figure 3

Immunoreactivity pattern of bone matrix proteins in human nondiseased aorta, intimal xanthoma, fibrous cap atheroma, and fibrocalcific plaques. The table represents the immuno-histochemical pattern of the bone matrix regulatory proteins MGP, OC, BSP, BMP-2, BMP-4, OPN, and ON in human atherogenesis. Fibrocalcific plaques were divided into cartilage tissue, calcium deposits, and bone tissue, structures that were present in these lesions. MGP, OC, and BSP were present in early as well as advanced lesions, whereas BMP-2, BMP-4, OPN, and ON were only present in advanced plaques. (Reproduced with permission from Dhore CR, et al. Arterioscler Thromb Vasc Biol. 2001;21:1998–2003.)

Figure 4

Coronary calcification and plaque morphology in humans. A is a radiograph of the coronary arteries following removal from the heart. B shows the type of radiographic calcification in different plaques. Radiographic calcification was typed according to the classification of Friedrich et al (Friedrich GJ, et al. Am Heart J 1994;128:435–41) and in brief absence of calcification, speckled, and fragmented (linear or wide, single focus of calcium >2 mm in diameter), or diffuse (≥5mm segment of continuous calcium). Bar graph in C shows mean area of calcification in different plaque morphologies in sudden coronary death victims. T-bars indicate SEM. AIT=adaptive intimal thickening; FA=fibroatheroma; LAD=left anterior descending artery; LCX=left circumflex artery; LD=left diagonal artery; LM=left main coronary artery; LOM=left obtuse marginal branch; PIT=pathologic intimal thickening; RCA=right coronary artery; TCFA=thin-cap fibroatheroma. (B and C are reproduced with permission from Burke AP, et al. Herz. 2001;26:239–244.)

Figure 5

Bar graphs showing total calcification score in sudden coronary death (SCD) victims stratified by decade in male and female (A) as well as in black and white (B). Calcification was scored based on one point per mm of epicardial artery involved by calcification by visual inspection of radiographs (density was not assessed). (A is reproduced with permission from Burke AP, et al. Z Kardiol 2000;89:Suppl 2,II/49-II/53. B is reproduced from Burke AP, et al. Circulation 2002;106:II-481)

Figure 6

A shows correlation between square root of coronary calcium area values (mm²) detected by histopathologic and microradiographic analysis and square root of plaque area values (mm2) for each of the 723 coronary artery segments in humans. B shows square root of coronary calcium area (mm²) detected by histopathologic and microradiographic analysis versus square root of lumen area (mm2) for each of the 723 coronary artery segments where no relation was identified. C shows relationship between percent stenosis and the degree of calcification in sudden coronary death victims. Each blue bar represents prevalence of any calcification (%), whereas each red dot represents mean calcification area (mm²). (A and B are reproduced with permission from Sangiorgi G, et al. J Am Coll Cardiol. 1998;31:126–133. Data in C is stratified by decades from Burke AP, et al. Herz. 2001;26:239–244)

Figure 7

Prevalence of various coronary plaque morphologies at 10% incremental cross-sectional area narrowing in sudden coronary death victims. Abbreviations as in Figure 4. (Data presented as each 10% increase in narrowing from Burke AP, et al. Herz. 2001;26:239–244)

Figure 8

Schematic illustrating four, non–mutually exclusive theories for vascular calcification: (1), cell death leading to release of apoptotic bodies and/or necrotic debris that may serve to nucleate apatite at sites of injury; (2), circulating nucleational complexes released from actively remodeling bone or matrix vesicular released locally; (3) loss of inhibition as a result of deficiency of constitutively expressed tissue-derived and circulating mineralization inhibitors leads to default apatite deposition; and (4) induction of bone formation resulting from altered differentiation of vascular smooth muscle or stem cells. (Reproduced and modified with permission from Speer MY, et al. Cardiovasc Pathol 2004;13: 63–70.)