William D. Wagner

A definition of the intima of human arteries and of its atherosclerosis-prone regions. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association.

T his report is a concise review of current knowledge of the structure and function of the intima of the aorta and the major distributing arteries. The main purpose of the review is to delineate normal arterial intima from atherosclerotic lesions and, in particular, to distinguish physiological adaptations from atherosclerotic increases in intimal thickness. To characterize normal intima, including the adaptive intimal thickenings, some of which represent locations in which atherosclerotic lesions are prone to develop, the structure, composition, and functions of the arterial intima in young people as well as in laboratory animals not subjected to known atherogenic stimuli are reviewed. This report on arterial intima is the first in a series of four. The second report will review and define initial, fatty streak, and intermediate types of atherosclerotic lesions, and the third report will review all types of advanced (i.e., potentially clinical and clinical) lesions. The overall objective is to define arterial intima and all types of atherosclerotic lesions, and then to postulate, in a fourth and final report, a valid and up-to-date pathobiological nomenclature and classification of atherosclerotic lesions.

A more sensitive assay discriminating galactosamine and glucosamine in mixtures.

Abstract A modification of a colorimetric assay (1) discriminating galactosamine and glucosamine in mixtures is presented. The procedure employs acetylation of the amino sugars at 25°C instead of at 0–1°C, resulting in five times the color intensity for galactosamine with no interference due to glucosamine. The assay of HCl hydrolysates of glycosaminoglycans and glycoproteins is possible in solutions that are less than 0.1 n .

In vitro and in vivo comparative colonization of Staphylococcus aureus and Staphylococcus epidermidis on orthopaedic implant materials

Clinically, Staphylococcus aureus appears to be the dominant organism associated with infected metal implants, whereas coagulase-negative staphylococcal strains are more frequently isolated from infected polymer implants. We reproduced this trend experimentally in vitro and in vivo. Discs of a titanium alloy, poly(methyl methacrylate) and ultra-high molecular weight polyethylene were exposed to a clinical isolate of Staphylococcus aureus or either of two strains of Staphylococcus epidermidis. Within 1 h Staphylococcus aureus was always the most rapid colonizer regardless of biomaterial. However, after 8 to 24 h, Staphylococcus aureus was present in higher numbers on metal and Staphylococcus epidermidis on polymers. Moreover, the exopolysaccharide produced by Staphylococcus epidermidis appeared to offer an effective protection against host defences in vivo.

Wound healing: A paradigm for lumen narrowing after arterial reconstruction

PURPOSE The intimal hyperplasia hypothesis that equates lumen narrowing after arterial injury with intimal mass has recently been challenged. Evidence has emerged to suggest that lumen narrowing is caused in large part by changes in artery wall geometry rather than intimal mass per se. We have begun to explore this hypothesis in a unique nonhuman primate model of atherosclerosis. METHODS Monkeys who were fed an atherogenic diet for 3 to 5 years underwent experimental angioplasty of the left iliac artery. The contralateral iliac artery served as an intraanimal control. Arteries were removed 2, 4, 7, 14, 28, or 112 days later for analysis (6 or 13 per time point). Angioplasty dilated arteries by fracturing atheroma and stretching or tearing the media. Cross-sections of injured arteries were analyzed for expression of extracellular matrix components and cell surface integrins that are important in wound healing. Antibodies, riboprobes, or histochemical stains specific for fibrin, hyaluronan, versican (chondroitin sulfate-containing proteoglycan), procollagen-I, elastin, and the alpha 2 beta 1 and alpha V beta 3 integrins were used. RESULTS A thin mural thrombus was seen at sites of denudation and plaque fracture (days 2 to 7). This provisional matrix was invaded by leukocytes (days 2 to 4) and alpha-actin-positive smooth muscle cells (SMCs; days 4 to 7). Thrombus was replaced by SMCs expressing hyaluronan and the associated versican proteoglycans (day 14). Versican was expressed throughout the neointima as it enlarged (day 28), but expression later subsided (day 112). Procollagen-I expression initially increased in the adventitia (day 4) and then in the forming neointima (day 14). Procollagen-I expression was found to persist within the adventitia and in the neointima in SMCs nearest the lumen (days 28 to 112). Elastin staining was prominent within the mature neointima (day 112) but not at earlier time points. Integrin expression also increased within the injured artery wall. alpha v beta 3 staining (fibrin[ogen] receptor) increased in the injured media (days 2 to 7) and was then seen throughout the early neointima (day 7). Low level expression of alpha V beta 3 subsequently persisted within the forming neointima (day 28). alpha 2 beta 1 (collagen receptor) expression increased in the neointima in SMCs nearest the lumen (day 28). CONCLUSIONS Lumen narrowing after angioplasty in this model of atherosclerosis is caused largely by decreased artery wall diameter. The pattern of matrix and integrin expression within the injured artery wall is in many ways analogous to that of healing wounds. These observations suggest that tissue contraction may play a role in lumen narrowing at sites of arterial reconstruction. Strategies to inhibit wound contraction may prove effective in preventing restenosis.

Molecular Interactions Leading to Lipoprotein Retention and the Initiation of Atherosclerosis

Atherosclerosis is distinguished by the accumulation of lipoprotein lipid within the arterial wall. An ionic interaction of positively charged regions of apolipoprotein (apo) B with matrix proteins, including proteoglycans, collagen, and fibronectin, is thought to initiate this process. Proteoglycans are complex glycoproteins containing highly negatively charged carbohydrate chains. These proteins are abundant in atherosclerosis lesions, and they associate with apoB-containing lipoproteins. Several specific regions of apoB may mediate this process. Other lipoprotein-associated proteins, including apoE and lipases, might also participate in this process. In addition, retention may occur via lipoprotein association with other matrix molecules or as a consequence of intra-arterial lipoprotein aggregation.

Hyaluronan enhances contraction of collagen by smooth muscle cells and adventitial fibroblasts: Role of CD44 and implications for constrictive remodeling.

Remodeling contributes to restenosis when cells shrink the artery wall at sites of injury. This may be analogous to wound healing, where tissue remodeling achieves wound contraction. Hyaluronan (HA) is prominent in wound matrix and inhibits fetal scarring. HA is also produced in the artery wall after angioplasty, where it may inhibit constrictive remodeling. This hypothesis was tested in vitro using a model of matrix contraction. Primate aortic smooth muscle cells and adventitial fibroblasts were seeded into collagen I gels containing increasing amounts of HA (0% to 50%, wt/wt). Both cell types reduced the diameter of collagen alone ≈65% at 18 hours. HA significantly increased gel contraction (diameter in mm: 0% HA, 7.7±0.9; 2%, 7.1±0.7; 10%, 6.7±0.5; 50%, 5.6±0.9;P <0.05 for ≥10%), cell spreading and telopodia, and pericellular accumulation of collagen fibrils. These effects were mediated in part by cellular HA binding, because an antibody against CD44 receptors blocked pericellular collagen accumulation and enhanced gel contraction without altering cell shape. The role of CD44 was specific, because inhibiting receptor for hyaluronic acid–mediated motility (RHAMM) had no effect. Blocking &bgr;1-integrins completely inhibited contraction of collagen, but gels containing HA required CD44 and &bgr;1-integrin blockade for complete inhibition. Enhanced collagen reorganization and contraction were not attributable to increased collagenase activity, because the metalloproteinase inhibitor batimastat had no effect. In summary, HA enhanced collagen reorganization by the cell types most likely to mediate constrictive remodeling after angioplasty. These effects were CD44-dependent, thus providing a potential target for therapies to prevent constrictive remodeling and restenosis.

Differentiated Macrophages Synthesize a Heparan Sulfate Proteoglycan and an Oversulfated Chondroitin Sulfate Proteoglycan That Bind Lipoprotein Lipase

Lipoprotein lipase (LpL), which facilitates lipoprotein uptake by macrophages, associates with the cell surface by binding to proteoglycans (PGs). Studies were designed to identify and characterize specific PGs that serve as receptors for LpL and to examine effects of cell differentiation on LpL binding. PG synthesis was examined by radiolabeling THP-1 monocytes and macrophages (a cell line originally derived from a patient with acute monocytic leukemia) with [35S]sodium sulfate and [3H]serine or [3H]glucosamine. Radiolabeled PGs isolated from the cell surface were purified by chromatography and identified as chondroitin-4-sulfate (CS) PG and heparan sulfate (HS) PG. A sixfold increase in CSPG and an 11-fold increase in HSPG accompanied cell differentiation. Whereas HS glycosaminoglycan chains from both monocytes and macrophages were 7.5 kD in size, CS chains increased in size from 17 kD to 36 kD with cell differentiation, and contained hexuronyl N-acetylgalactosamine-4,6-di-O sulfate disaccharides. LpL binding was sevenfold higher to differentiated cells, and affinity chromatography demonstrated that two cell surface PGs bound to LpL: HSPG and the oversulfated CSPG produced only by differentiated cells. We conclude that differentiation-associated changes in cell surface PG of human macrophages have functional consequences that could increase the atherogenic potential of the cells.

The development of pigeon strains with selected atherosclerosis characteristics

Abstract Parental pairs of White Carneau, Show Racer, and F 2 (progeny of F 1 crosses between White Carneau and Show Racer) pigeons were identified according to specific features of the atherosclerosis of their offspring. Progeny were evaluated for atherosclerotic variables after being fed a diet containing 0.5% cholesterol for 6 mo. No significant differences were seen in the degree of hypercholesterolemia between breeds; however, within breeds all males had significantly higher serum cholesterol concentrations than females. Aortic atherosclerosis was more severe in White Carneau than in Show Racer or F 2 with F 2 pigeons being intermediate in the response. All autopsied progeny dying during the experiment showed more aortic and coronary atherosclerosis than their surviving sibs. Likewise, there was more coronary and aortic atherosclerosis in pigeons with myocardial infarcts found when they were killed at the end of the experiment. Aortic root and coronary artery atherosclerosis were assessed as important causative factors in myocardial infarction. Based on the atherosclerotic characteristics of progeny, the following strains have been selected. 1. 1. A strain of White Carneau with severe aortic atherosclerosis having mean aortic cholesterol concentrations of 60.42 mg/g aorta. 2. 2. A strain of Show Racer with very little aortic atherosclerosis having a mean aortic cholesterol concentration of 2.40 mg/g aorta. 3. 3. A strain of F 2 with concomitant severe coronary artery atherosclerosis (mean coronary artery index of 38) and very little aortic atherosclerosis (mean aortic cholesterol concentration of 6.13 mg/g aorta). 4. 4. A Show Racer and an F 2 strain with a frequency of myocardial infarction of 43 and 60%, respectively.

Comparative primate atherosclerosis: I. Tissue Cholesterol Concentration and Pathologic Anatomy

Abstract Stump-tailed macaques ( Macaca arctoides ), African green monkeys ( Cercopithecus aethiops ), squirrel monkeys ( Saimiri sciureus ), and woolly monkeys ( Lagothrix lagothricha ) were fed control, solid atherogenic (1 mg cholesterol/cal) or liquid diets containing 0, 0.5, or 1 mg cholesterol/cal. Stump-tailed macaques fed the solid atherogenic diet had the highest tissue and serum cholesterol concentration (about 700 mg/dl) and the most extensive atherosclerosis. These monkeys appeared to respond differently to diets containing 1 mg cholesterol/cal. Those animals fed the liquid diet had higher liver cholesterol concentration but lower serum cholesterol concentration than animals fed the solid diet. African green monkeys fed the solid atherogenic diet had serum cholesterol concentrations of about 450 mg/dl. A greater percentage of the abdominal aorta was covered by plaque than the thoracic aorta. Coronary artery atherosclerosis was focal with the largest plaques being found in the left main coronary artery. The microscopic appearance of these plaques was similar to that of plaques from people. Squirrel monkeys fed the atherogenic diet were the most variable group. The average serum cholesterol concentration averaged about 450 mg/dl (range: 291 to 716). The percentage of aorta covered by plaque ranged from 0 to 55% with more thoracic than abdominal aortic atherosclerosis. There were findings consistent with hemorrhage in plaques from two animals. These monkeys, like stump-tailed macaques but unlike African green monkeys had relatively high liver cholesterol concentrations. Woolly monkeys appeared to develop atherosclerosis when fed 1 mg cholesterol/cal but did not have greatly elevated serum cholesterol concentrations.

Plasma low density lipoprotein accumulation in aortas of hypercholesterolemic swine correlates with modifications in aortic glycosaminoglycan composition

Arterial wall sulfated glycosaminoglycans (GAG) of matrix proteoglycans have been implicated in the retention of plasma low density lipoproteins in the early stages of atherosclerosis. We have studied modifications in porcine aortic GAG composition after 4 and 11 weeks of diet-induced hypercholesterolemia. After these time intervals no grossly visible atherosclerotic lesions were discerned. GAG changes were correlated with tissue LDL accumulation estimated by quantification of immunochemically-identifiable apolipoprotein B (apoB). Values of apoB ranged from less than 10 to 250 ng/mg wet weight of aorta, and correlated significantly with tissue total cholesterol contents. Although total GAG concentrations did not differ between a normolipemic control and the two diet groups, apoB showed a significantly positive correlation with the percent of total GAG that was chondroitin sulfate and a significantly negative correlation with the percent of total GAG that was dermatan sulfate. Total tissue cholesterol likewise demonstrated similar correlations with GAG. Since areas of the aorta were chosen that were devoid of intimal thickening, these metabolic changes may occur in the inner part of the arterial tunica media. The results suggest that the accumulation of plasma LDL in the arterial wall following hypercholesterolemia may induce alterations in arterial GAG composition, presumably by affecting GAG synthesis by medial smooth muscle cells.