Arlen R. Severson

Arterial Calcification and Not Lumen Stenosis Is Highly Correlated With Atherosclerotic Plaque Burden in Humans: A Histologic Study of 723 Coronary Artery Segments Using Nondecalcifying Methodology

OBJECTIVES This study was designed to evaluate whether calcium deposition in the coronary arteries is related to atherosclerotic plaque burden and narrowing of the arterial lumen. BACKGROUND Many studies have recently documented the feasibility of electron beam computed tomography to detect and quantify coronary artery calcification in patients. Although these studies suggest a general relation between calcification and severity of coronary artery disease, the value of coronary calcium in defining atherosclerotic plaque and coronary lumen narrowing is unclear. Previous pathologic comparisons have failed to detail such a relation in identical histologic sections. This finding may be due to atherosclerotic remodeling. METHODS A total of 37 nondecalcified coronary arteries were processed, sectioned at 3-mm intervals (723 sections) and evaluated by computer planimetry and densitometry. RESULTS A significant relation between calcium area and plaque area was found on a per-heart basis (n = 13, r = 0.87, p < 0.0001), per-artery basis (left anterior descending coronary artery [LAD]: n = 13, r = 0.89, p < 0.0001; left circumflex coronary artery [LCx]: n = 11, r = 0.7, p < 0.001; right coronary artery [RCA]: n = 13, r = 0.89, p < 0.0001) and per-segment basis (n = 723, r = 0.52, p < 0.0001). In contrast, a poor relation existed between residual histologic lumen area and calcium area for individual hearts (r = 0.48, p = NS), individual coronary arteries (LAD: r = 0.59, p = NS; LCx: r = 0.10, p = NS; RCA: r = 0.59, p = NS) and coronary segments (r = 0.07, p = NS). Longitudinal changes in external elastic lamina areas were highly correlated with changes in plaque area values (r = 0.60, p < 0.0001), whereas lumen area did not correlate with plaque size change (r = 0.01, p = NS). CONCLUSIONS Coronary calcium quantification is an excellent method of assessing atherosclerotic plaque presence at individual artery sites. Moreover, the amount of calcium correlates with the overall magnitude of atherosclerotic plaque burden. This study suggests that the remodeling phenomenon is the likely explanation for the lack of a good predictive value between lumen narrowing and quantification of mural calcification.

Diffuse calcification in human coronary arteries. Association of osteopontin with atherosclerosis.

Coronary atherosclerosis is frequently associated with calcification of arterial plaque. To understand the mechanisms responsible for the formation of atherosclerotic calcification, we examined human coronary arteries for the presence and extent of mineral. In sections stained specifically for mineral, staining was diffuse and present in all atherosclerotic plaques. Hydroxyapatite was not detected in normal coronary artery sections. Distribution of hydroxyapatite coincided with a similar distribution of calcium detected by a radiodense pattern using contact microradiography of the same sections before cytochemical staining. By energy-dispersive x-ray microanalysis, the chemical composition of calcified sites was identical to hydroxyapatite (Ca10[PO4]6[OH]2), the major inorganic component of bone. Osteopontin is a phosphorylated glycoprotein with known involvement in the formation and calcification of bone and is regulated by local cytokines. Human coronary artery segments (14 normal and 34 atherosclerotic) obtained at autopsy were evaluated immunohistochemically using polyclonal antibodies generated against human osteopontin. Immunohistochemistry for osteopontin indicated intense, highly specific staining in the outer margins of all diseased segments at each calcification front; staining was evident throughout the entire plaque. Conversely, arterial segments free of atheroma and calcification and sections treated with nonimmune serum had no evidence of positive staining. Osteopontin, a protein involved in mineralization is specifically associated with calcific coronary atheroma and may play an important role in the onset and progression of this disease in human coronary arteries. The deposition of noncollagenous proteins such as osteopontin may regulate the presence or absence of calcification and ultimately alter vessel compliance.

Matrix proteins associated with bone calcification are present in human vascular smooth muscle cells grown in vitro

SummaryAtherosclerotic lesions are composed of cellular elements that have migrated from the vessel lumen and wall to form the cellular component of the developing plaque. The cellular elements are influenced by various growth-regulatory molecules, cytokines, chemoattractants, and vasoregulatory molecules that regulate the synthesis of the extracellular matrix composing the plaque. Because vascular smooth muscle cells (VSMC) constitute the major cellular elements of the atherosclerotic plaque and are thought to be responsible for the extracellular matrix that becomes calcified in mature plaques, immunostaining for collagenous and noncollagenous proteins typically associated with bone matrix was conducted on VSMC grownin vitro. VSMC obtained from human aorta were grown in chambers on glass slides and immunostained for procollagen type I, bone sialoprotein, osteonectin, osteocalcin, osteopontin, decorin, and biglycan. VSMC demonstrated an intense staining for procollagen type I, and a moderately intense staining for the noncollagenous proteins, bone sialoprotein and osteonectin, two proteins closely associated with bone mineralization. Minimal immunostaining was noted for osteocalcin, osteopontin, decorin, and biglycan. The presence in VSMC of collagenous and noncollagenous proteins associated with bone mineralization suggest that the smooth muscle cells in the developing atherosclerotic plaque play an important role in the deposition of the extracellular matrix involved in calcification of developing lesions.

Studies of the metabolism of separated bone cells - I. Techniques of separation and identification

The mechanical separation of rat skeletal tissue into viable populations of periosteal cells, osteoblasts, osteocytes, and marrow cells has been undertaken. Periosteal cells and osteocytes were obtained as tissue preparations, while osteoblasts and marrow cells were isolated as individual cells. The cell populations were identified during the preparative procedure by their histological and histochemical appearance. Viability of these preparations was demonstrated by their metabolic activity during short-term incubations and their histological appearance and vital staining before and after such incubations. The cell populations produced carbon dioxide and lactate, consumed oxygen, and incorporated uridine into RNA. Comparison of uridine incorporation into RNA of cells from control and parathyroid hormone treated rats was compatible with a differential effect of parathyroid hormone on the cell populations. These findings indicate that the technique described can be utilized to produce viable bone cells of different types, and may offer a new means for characterizing the role of each cell type in the metabolism of skeletal tissue.RésuméLa séparation mécanique du tissu squelettique du rat en cellules périostées, en ostéoblastes, en ostéocytes et en cellules de la moelle a été réalisée. Les cellules périostées et les ostéocytes ont été obtenues sous forme de préparations tissulaires, alors que les cellules ostéoblastiques et celles de la moelle ont été isolées sous forme de cellules individuelles. Les populations cellulaires sont identifiées par leurs caractères histologiques et histochimiques. La vitalité de ces préparations est věrifiée par leur activité métabolique pendant des incubations de courte durée et des examens. histologiques et des colorations vitales, réalisés avant et après incubation. Les cellules produisent du gaz carbonique et du lactate, consomment de l’oxygène et incorporent l’uridine en RNA. La comparaison de l’incorporation d’uridine en RNA de cellules de rats témoins et de rats traités par hormone parathyroïdienne concorde avec une action différentielle de la parathormone sur les divers types de cellules. Il semble que la technique décrite permette de produire des cellules osseuses vivantes de divers types et fournit ainsi le moyen d’élucider le rôle métabolique de chaque type cellulaire du squelette.ZusammenfassungDie mechanische Trennung von Rattenskelett-Gewebe in lebensfähige Populationen von Periost-Zellen, Osteoblasten, Osteozyten und Markzellen ist unternommen worden. Die Periostzellen und Osteozyten wurden als Gewebepräparate erhalten, während die Osteoblasten und Markzellen als individuelle Zellen isolier wurden. Die Zell-Populationen wurden während des Herstellungsverfahrens histologisch und histochemisch identifiziert. Die Lebensfähigkeit dieser Präparate wurde festgestellt durch ihre metabolishe Aktivität während Kurzzeit-Inkubationen und durch ihr histologisches Aussehen sowie durch Vitalfärbung vor und nach solchen Inkubationen. Die Zellpopulationen produzierten Kohlendioxyd und Lactat, verbrauchten Sauerstoff und bauten Uridin in RNS ein. Der Vergleich zwischen Uridineinbau in RNS von Zellen aus Kontrolltieren und aus mit Parathormon behandelten Ratten war mit der Differentialwirkung von Parathormon auf die Zellpopulationen vereinbar. Diese Befunde deuten darauf hin, daß die beschriebene Technik benützt werden kann, um verschiedene Typen von lebensfähigen Knochenzellen zu produzieren und daß sie einen neuen Weg öffnen kann, um die Rolle jedes Zelltyps im Stoffwechsel des Skelettgewebes zu charakterisieren.

Aluminum toxicity perturbs long bone calcification in the embryonic chick.

Abstract Long bone calcification in chick embryos acutely- or chronically-treated with aluminum (Al) citrate was investigated. Acutely treated embryos received 100 μl of 60 mM Al citrate, 60 mM sodium (Na) citrate, or 0.7% sodium chloride on day 8 of incubation. Chronically treated embryos received a daily 25 μl dose of the above solutions beginning on day 8. Following 2–8 days of additional incubation, blood was collected, embryos killed, hind limbs radiographed, and tibias collected. Radiography indicated that Al administration resulted in a persistent angulation in the mid-diaphysis of tibias and femurs and a transient mineralization defect during the 10- to 12-day period of incubation. Tibias from 10- to 12-day embryos which were administered Al contained significantly less (P < 0.005) bone calcium (Ca) compared with tibias from NaCl-treated embryos. By day 14 there were no significant differences among the Ca content of tibias from embryos acutely treated with Al citrate, Na citrate or NaCl. Similarly, the rate of 45Ca uptake by tibias of embryos treated with Al was significantly lower on days 10 (acute) and 12 (chronic) with no significant differences in Ca uptake rate among the three treatment groups by day 16. In each treatment group bone alkaline phosphatase (ALPase) activity increased approximately tenfold between days 10 and 16. At all stages, bone ALPase activity was consistently higher and significantly different (chronic) compared with levels in NaCl-treated embryos. In contrast, Al had no significant effect on the rate of tibia collagen and noncollagenous protein synthesis or serum levels of procollagen carboxy-terminal propeptide (PICP), osteocalcin, and parathyroid hormone (PTH).

Influence of short-term aluminum exposure on demineralized bone matrix induced bone formation

The effects of aluminum exposure on bone formation employing the demineralized bone matrix (DBM) induced bone development model were studied using 4-week-old Sprague-Dawley rats injected with a saline (control) or an aluminum chloride (experimental) solution. After 2 weeks of aluminum treatment, 20-mg portions of rat DBM were implanted subcutaneously on each side in the thoracic region of the control and experimental rats. Animals were killed 7, 12, or 21 days after implantation of the DBM and the developing plaques removed. No morphological, histochemical, or biochemical differences were apparent between plaques from day 7 control and experimental rats. Plaques from day 12 control and experimental rats exhibited cartilage formation and alkaline phosphatase activity localized in osteochondrogenic cells, chondrocytes, osteoblasts, and extracellular matrix. Unlike the plaques from control rats that contained many osteoblastic mineralizing fronts, the plaques from the 12-day experimental group had a preponderance of cartilaginous tissue, no evidence of mineralization, increased levels of alkaline phosphatase activity, and a reduced calcium content. Plaques developing for 21 days in control animals demonstrated extensive new bone formation and bone marrow development, while those in the experimental rats demonstrated unmineralized osteoid-like matrix with poorly developed bone marrow. Alkaline phosphatase activity of the plaques continued to remain high on day 21 for the control and experimental groups. Calcium levels were significantly reduced in the experimental group. These biochemical changes correlated with histochemical reductions in bone calcification. Thus, aluminum administration to rats appears to alter the differentiation and calcification of developing cartilage and bone in the DBM-induced bone formation model and suggests that aluminum by some mechanism alters the matrix calcification in growing bones.

Differentiation of Mononuclear Cells into Multinucleated Osteoclast-Like Cells

Experimental evidence accumulated in recent years suggests that the osteoclast is derived from the fusion of mononuclear precursors which are of hematopoietic origin. Mononuclear cells were isolated from the spleen and bone marrow of young rats in order to examine osteoclast formation. The isolated cells were placed in diffusion chambers containing devitalized bone fragments freed of soft tissue, and the chambers sealed and placed in the peritoneal cavity of host rats. The host animals were killed after 4 days, and the bone removed from the chambers for examination. Light-microscopic examination demonstrated two types of cells adjacent to the bone surface, one a flattened and elongated mononuclear cell, and the other a larger and frequently multinucleated cell which had the morphological appearance of an osteoclast. Scanning electron microscopy demonstrated numerous flattened and elongated cells adjacent to the bone surface, as well as a second cell type which had dorsal membrane specializations and numerous lateral microprojections attaching to the bone surface. The second cell type was thought to correspond to the osteoclast-like cells seen with light microscopy. The observations suggest that osteoclast-like cells differentiate from mononuclear precursor cells of hematopoietic tissue.

Aluminum effects on blood chemistry and long bone development in the chick embryo

Body growth, blood chemistry, and long bone development of 10- to 16-day chick embryos (Gallus gallus) treated with aluminum (Al) citrate, sodium (Na) citrate, or sodium chloride (NaCl) were investigated. Two administration protocols were used. Acutely-treated embryos received 6.0 μmol Al citrate or Na citrate on day 8 of incubation. Chronically-treated embryos received a daily dose of 1.5 μmol Al citrate or Na citrate beginning on day 8 of incubation. For both protocols, Al citrate and Na citrate had no significant influence on viability or body weight. Al citrate-treated embryos had: (a) significantly shorter mean tibia lengths by day 16 of incubation, (b) a consistently lower ratio of tibia length: body weight on all days investigated, and (c) a persistent mid-diaphyseal malformation (angulation) of the femur and tibia. Spatially correlated with the malformation was a calcification defect detected by alizarin red S staining of intact tibias and the accumulation of aluminum as demonstrated by acid solochrome azurine staining of histological sections. Aluminum was localized at the mineralization front of the osteogenic collar surrounding the cartilage core of the tibia. Aluminum citrate or Na citrate had no significant effect on serum total calcium, inorganic phosphorus, total alkaline phosphatase activity, or creatinine, except for a transitory hypercalcemia (day 10) and phosphatemia (days 10 and 12) in Al citrate-treated embryos. The concomitant localization of Al and the early calcification defect in the region of tibial malformation implicate aluminum in the pathogenesis of the skeletal abnormality.

Histochemical Demonstration of Adenosine Triphosphatase in Dental Tissue

Limited attention has been given to the histochemical demonstration of nucleoside phosphatases in dental tissue (M. BURSTONE, Anni NY Acad Sci 85:431-444, 1960; W. GIBSON and H. FULLMLR, Periodontics 5:226-232, 1967). Severson El AL (J Histochein Cvtoc/zemi 15:550-552, 1967) have shown that adenosine triphosphatase (ATPase) activity can be localized in skeletal cells. The present report is of preliminary observations of ATP hydrolysis in association with cells of the mouse incisor. The mandibles of young mice were removed immediately after they were killed, freed of excess soft tissue, separated into right and left halves, and demineralized for 72 hours in 10% aqueous disodium ethylenediaminetetraacetate (EDTA), pH 7.2, at 4C (K. BALOGH, J Histochem Cvtochem 10:232, 1962). After deemineralization, the tissues were frozen on a block of solid CO2, attached to a chuck, and cut at 10i in a cryostat. The frozen sections were placed on cold albumenized slides, oriented, thawed, dried in a cool air stream, and fixed for ten minutes in cold ( -2C to -3C) 10% formalin, buffered with sodium acetate, and adjusted to pH 7.2. The sections were washed, treated with 10% EDTA for 20 minutes at room temperature to ensure complete demineralization, rewashed, and incubated for 30 minutes at 37C in the lead-ATP medium of Wachstein and Meisel (Amer J Clitz Pat/i 27: 13-23, 1957). After the usual treatment with (NH4,)S, the sections were postfixed in 10% neueral buffered formalin, dehydrated, cleared, and mounted. Preliminary evaluation of the specificity of the histochemical reaction was undertaken by omitting ATP from the medium and substituting an equimolar concentration of beta-glycerophosphate. Additional sections were treated for ten minutes in distilled water at 90C prior to ATP-substrate incubation. The enzyme activity at the level visible with a light microscope was localized primarily within the cells, and there was minimal evidence of diffusion. Sections were unstained when ATP was omitted from the incubating medium or substituted for by beta-glycerophosphate. Heat treatment of sections eliminated the ATPase reaction. Ameloblasts and cells of the underlying stratum intermedium were prominent (lllustra-

Application and evaluation of the lead-adenosine triphosphatase method in skeletal tissue

Application and evaluation of the lead-ATPase histochemical method in skeletal tissue has demonstrated an intracellular localization of enzyme activity. The skeletal tissue was demineralized for 72 hr in cold 10% aqueous EDTA adjusted to pH 7.2. Frozen sections were cut and placed on cold albumenized slides, oriented, thawed, dried in a cool air stream, and fixed for 10 min in cold (−2 to −3 C) 10% formalin buffered with Na-acetate and adjusted to pH 7.2. The sections were washed, treated with 10% EDTA for 20 min at room temperature, rewashed, and incubated for an optimal period of 30 min at 37 C. in the lead-ATP medium of Wachstein and Meisel. Following incubation the sections were washed, treated for 1 min with 1% (NH4)2S, rewashed, immersed for 30 min in 10% buffered formalin, dehydrated, cleared, and mounted. Evaluation of the substrate specificity suggests that other phosphatases associated with skeletal tissue do not complicate the ATPase reaction.