College of American Pathologists-Centers for Disease Control collaborative study for evaluating reference materials for total serum cholesterol measurements.

Several recent studies to evaluate the performance of laboratory instruments have shown that with some instrument systems processed (lyophilized, frozen, and stabilized) materials exhibit matrix effects that cause the assay for cholesterol to respond differently for them than for patient specimens. To understand this phenomenon better the College of American Pathologists, Northfield, III, and the Centers for Disease Control, Atlanta, Ga, have conducted a collaborative study with 44 laboratories where 16 instruments manufactured by nine companies are evaluated. The purposes were to assess measurement variation on several reference materials used for standardizing total cholesterol measurements and to evaluate a new stabilized liquid serum as a potential reference material. Lypophilized, frozen, fresh-frozen, and stabilized materials at three concentrations were measured for total cholesterol. The results show that the average coefficient of variation of measured total cholesterol for all instruments, laboratories, vials, and replicates is 3.6% to 4.1% for each of the materials measured (excluding the results for one instrument). For one instrument, however, significant bias was found on the stabilized liquid serum material. Results from the fresh-frozen materials indicate that the instrument systems evaluated allow laboratories to attain the National Cholesterol Education Program analytical performance goals.

Influence of hypercholesterolemia and cholesterol accumulation on rabbit carrageenan granuloma macrophage activation.

These experiments were designed to determine whether hypercholesterolemia and the accumulation of cholesterol or cholesteryl esters in rabbit carrageenan granuloma macrophages might influence selected markers of macrophage activation. Granulomas induced by subcutaneous injection of carrageenan into rabbits were harvested after 4, 14, and 28 days. Macrophages were isolated from granuloma tissues by collagenase digestion and cultured overnight. Secretion of lysosomal beta-glucuronidase, membrane 5'-nucleotidase, cellular plasminogen activator, and superoxide anion generation were measured as markers of activation. beta-Glucuronidase activity secreted into the media by granuloma macrophages from normocholesterolemic (NC) and hypercholesterolemic (HC) rabbits showed a trend toward an increase with time between 4 and 14 days in both groups. This was confirmed in a separate experiment with a significant increase by 14 days, together with a significantly greater secretion by NC macrophages and a significantly elevated level of cellular beta-glucuronidase activity in NC relative to HC macrophages. Activity of the membrane ectoenzyme 5'-nucleotidase was minimal in lysates of NC or HC macrophages, in contrast to freshly isolated human monocytes, indicating that both NC and HC granuloma macrophages were highly activated. Cellular plasminogen activator activity was significantly increased between 4 and 14 days, and was significantly greater in HC than in NC macrophages at 14 days. Stimulation of macrophages with phorbol myristate acetate increased superoxide anion generation by both NC and HC macrophages; however, no difference in superoxide anion generation was observed between macrophages from NC and HC rabbits. On the basis of the 5'-nucleotidase findings, it is concluded that both the NC and HC granuloma macrophages are highly activated, and further that hypercholesterolemia does not enhance macrophage generation of superoxide anion, either spontaneously or as the result of phorbol myristate acetate stimulation. Although hypercholesterolemia results in macrophage activation in terms of an increased cellular plasminogen activator activity, the secretion of the lysosomal enzyme beta-glucuronidase is diminished. Thus, hypercholesterolemia associated with macrophage cholesterol and cholesteryl ester accumulation has no consistent overall influence on activation, a finding of potential importance in the context of atherogenesis.

Low-density lipoprotein endocytosis. II. Influence of the multivalent ligand cationized ferritin on acetylated low-density lipoprotein endocytosis in cultured cells.

Interactions of the basic multivalent ligand cationized ferritin (CF) with cultured cells markedly alter their endocytic function. In this study, the influence of CF treatment on the binding, internalization, and degradation of chemically modified (acetylated) low-density lipoproteins (Ac-LDL) was examined in aortic smooth muscle cells (SMC); and in normal and FH mutant LDL receptor-negative human skin fibroblasts, which lack the Ac-LDL (scavenger) receptor; and in vascular endothelial cells, which normally express the receptor. Although CF treatment of all three cell types at 37 degrees C resulted in the induction of Pronasesensitive, high-capacity, high-affinity binding (Kd = 12.0 +/- 2.0 nM at 4 degrees C) of labeled Ac-LDL, which at 37 degrees C was accompanied by significant internalization and degradation, these processes were not receptor-mediated. CF-induced high-affinity binding was inhibited by unlabeled Ac-LDL, fucoidan, carrageenan, and dextran sulfate but was unaffected by native LDL and albumin and only partially inhibited by acetylated albumin. However, analysis of membrane preparations of the cells for "scavenger" receptor protein by solid-phase filtration assay and Western blotting identified the receptor in endothelial cells and in granuloma (positive control) macrophages, but not in either CF-treated or untreated SMC. In addition, studies with both glutaraldehyde-fixed cells and CF bound to culture dishes indicated that Ac-LDL avidly binds to CF. Further, ultrastructural studies using colloidal gold-conjugated Ac-LDL showed Ac-LDL preferentially binding to CF aggregates on the cell surface. Thus, these studies indicate that treatment of cells with CF induces an endocytic process which, although remarkably similar to the scavenger pathway, is mediated by Ac-LDL binding to membrane-associated CF. These observations have implications in terms of mechanisms that might regulate the endocytosis of modified low-density lipoproteins.

Influence of the acyl-CoA: cholesterol O-acyltransferase inhibitor, CL 277082, on cholesteryl ester accumulation in rabbit macrophage-rich granulomas and hepatic tissue.

The influence of the acyl-CoA: cholesterol O-acyltransferase (ACAT) inhibitor, CL 277082, on macrophage cholesteryl ester accumulation in a rabbit carrageenan granuloma macrophage-foam cell model was studied. Diets were supplemented with 0.3% cholesterol and 6% peanut oil with or without the inhibitor (0.25%) for 4 weeks prior to granuloma induction, and macrophage-rich granuloma tissue was harvested 14 days after carrageenan injection. Serum cholesterol was monitored biweekly, and plasma lipoproteins were isolated terminally. Total, free and esterified cholesterol contents were measured in hepatic and granuloma tissue. In hepatic tissue, administration of CL 277082 resulted in an 80% reduction in the content of total cholesterol, a 37% decrease in free cholesterol, and a 90% decrease in esterified cholesterol. Similarly, in macrophage-rich granuloma tissue, total cholesterol content was decreased by 44%, and esterified cholesterol content by 61%, with no change in free cholesterol. Additionally, CL 277082 was shown to inhibit granuloma tissue ACAT activity by 45%, VLDL mass was decreased slightly, LDL mass increased 3.4-fold and HDL mass was similar in both the inhibitor-treated and control animals. CL 277082 resulted in a 57% decrease in VLDL cholesteryl ester content and a 4.5-fold increase in triacylglycerol. Cholesteryl ester content in LDL was decreased by 31% and LDL triacylglycerol was increased 5.2-fold, while the only change in HDL composition was a 3.5-fold increase in triacylglycerol. The reductions in both hepatic tissue and macrophage-rich granuloma tissue esterified cholesterol accumulation are considered to be due largely to cellular ACAT inhibition, and the altered distribution and composition of the plasma lipoproteins.

Activation of human peripheral blood monocytes by lipoproteins.

Activation of human peripheral blood monocytes could enhance their attachment and or migration into the arterial intima and their various secretory and other functions, thus influencing the pathogenesis of atherosclerosis. In these experiments the authors have explored the role of lipoproteins in the activation of human blood monocytes. Monocytes were purified from citrated blood by Histopaque density gradient centrifugation and countercurrent centrifugal elutriation and cultured in DMEM in the presence of 20% acid-treated autologous serum or 100 micrograms/ml each of VLDL, LDL, Ac-LDL, and HDL. Secretion of beta-glucuronidase activity into the media was measured as a marker of activation. All of the lipoprotein density classes as well as serum stimulated secretion of beta-glucuronidase activity, with LDL and Ac-LDL having a greater influence than serum, VLDL, or HDL. Serum and LDL also stimulated secretion of prostaglandin E into the culture medium. Incubation of monocytes with serum or LDL in the presence of inhibitors of arachidonate metabolism (NDGA and indomethacin) resulted in a significant decrease in secreted and intracellular beta-glucuronidase activity, indicating a role for products of arachidonate metabolism in the activation of monocytes by lipoproteins.

Activation of human blood monocytes by adherence to tissue culture plastic surfaces.

Human monocytes, purified by countercurrent centrifugal elutriation, were cultured either in plastic dishes or in Teflon vials to determine if attachment would result in activation. beta-Glucuronidase activity, 5'-nucleotidase activity, plasminogen activator, and superoxide anion generation were measured as markers of monocyte activation. Conditioned media and cell lysates were assayed at 2, 4, 8, and 10 hr and then daily for 6 days. Monocytes cultured in plastic dishes secreted a significantly greater proportion of their beta-glucuronidase into the medium than those cultured in Teflon vials. The activity of 5'-nucleotidase was lower in monocytes cultured in plastic dishes, consistent with greater activation. Cellular plasminogen activator levels and the capacity for superoxide anion generation were enhanced in cells cultured in plastic dishes, relative to monocytes cultured in Teflon vials. These observations indicate that monocyte attachment in plastic surfaces results in their activation, a phenomenon that may influence the nature and interpretation of experimental data derived from cultured adherent monocytes or macrophages.

Monocyte-macrophage participation in atherogenesis: inflammatory components of pathogenesis.

This article has addressed the roles of the monocyte-macrophage in atherogenesis and factors influencing monocyte recruitment to the intima. The diversity of the secretory products of the macrophage and their putative participatory roles in pathogenesis have been reviewed and discussed. Additionally, we have presented summary data on the monocyte chemoattractant peptide SMC-CF and on the differentiation of the monocyte-derived foam cell. Discussion has centered on the concept of atherosclerosis as an inflammatory process.

Stimulation of albumin endocytosis by cationized ferritin in cultured aortic smooth muscle cells.

Anionic microdomains within the aortic smooth muscle cell (SMC) surface glycocalyx represent a potential barrier to the endocytosis of anionic plasma proteins. Cultured SMCs exposed briefly to cationized ferritin (CF) exhibit ultrastructural aggregations of surface anionic sites resulting in intervening areas essentially devoid of anionic charge. Preincubation of cultured aortic medial SMCs with 0.2 mg/ml CF for 1 minute at 37 C resulted in a 4-fold increase in binding and a 13-fold increase in internalization of 125I-human serum albumin (125I-HSA) relative to cells pretreated with native ferritin. When both the CF preincubation and the endocytosis were performed at 4 C, the influence of CF was abolished. Studies at 4 C indicated that CF pretreatment of SMC at 37 C induced high affinity (Kd = 1.5 nM) saturable 125I-HSA binding, in addition to low-affinity nonsaturable binding. These results were further confirmed by binding competition studies using increasing concentrations of unlabeled HSA. In contrast, low-density lipoprotein, a large anionic molecule, failed to compete with 125I-HSA for binding sites on CF-pretreated SMCs at either 4 or 37 C. Pulse-chase studies at 37 C indicated that 20-30% of internalized 125I-HSA was degraded, and 40-50% exocytosed within 24 hours in CF-treated cells. CF pretreatment of the SMCs did not significantly enhance the uptake of 14C-sucrose as a measure of fluid-phase endocytosis at 30 and 60 minutes. The results of these studies emphasize the potentially important regulatory roles of cell-surface anionic charge distribution and cationic molecules in cellular endocytosis.

Evolution of foam cells in subcutaneous rabbit carrageenan granulomas. II. Tissue and macrophage lipid composition.

This study describes the lipid composition of differentiating macrophage-derived foam cells in the inflammatory carrageenan granuloma. In this model, macrophages exposed in vivo to diet-induced hypercholesterolemia progressively accumulate electron-translucent lipid inclusions; and at 14 and 28 days, many assume the morphologic features of arterial plaque foam cells. Subcutaneous carrageenan granulomas were induced in 24 pellet-fed (NC) and 24 cholesterol-fed (HC) rabbits, and tissue was harvested at 4, 14, and 28 days. Total (TC) and free cholesterol (FC), cholesteryl esters (CEs), CE fatty acids, triglycerides (TGs), and phospholipids (PLs) were measured on lipid extracts from tissue. TC, FC, and CEs were also measured on isolated, cultured granuloma macrophages. Tissue TCs and FCs were significantly elevated in HC relative to NC rabbits at both 14 and 28 days (P less than 0.005 and P less than 0.01, respectively). CE accumulation in HC granuloma tissue was 80-fold greater at 14 days and 178-fold greater at 28 days (P less than 0.005), compared with NC granulomas. Oleic acid (18:1), the principal CE fatty acid in both NC and HC granulomas, accounted for significantly more (P less than 0.05) of the total CE fatty acids in HC (48%) relative to NC granulomas (37%). No net accumulation of TG was observed with time in NC or HC animals. Although diet did not influence tissue PL content, significant increases (P less than 0.05) were observed at 14 days in NC rabbits and at 14 and 28 days in HC rabbits relative to 4-day levels. CE accumulation was significantly greater in cultured macrophages isolated from HC granulomas at 14 days (P less than 0.001) and 28 days (P less than 0.01). These findings have demonstrated the significant accumulation of CEs in both HC granuloma tissue and in cultured HC macrophage/foam cells in vivo. The carrageenan granuloma model has, we believe, considerable potential for defining mechanisms responsible for CE accumulation in the differentiating macrophage-derived foam cell.

Atherosclerosis as an inflammatory process. The roles of the monocyte-macrophage.

In this brief review, we have addressed the roles of the monocyte-macrophage in atherogenesis, with emphasis on the recruitment to the arterial wall. We have presented summary data on the SMC-derived chemoattractant protein and also on the temporal evolution of monocyte-derived macrophages into cholesteryl ester-rich foam cells. The concept of atheroma as an inflammatory process has also been discussed.

Evolution of foam cells in subcutaneous rabbit carrageenan granulomas: I. Light-microscopic and ultrastructural study.

With an increasing interest in the role of the monocyte-macrophage in the pathogenesis of atherosclerosis and as a progenitor of plaque intimal foam cells, a model for the study of foam-cell differentiation in an extravascular environment has been developed. Granulomas were induced in 25 normocholesterolemic (NC) and 28 hypercholesterolemic (HC) rabbits by the subcutaneous injection of 15 ml of 1% carrageenan. Granuloma tissue was harvested at 4, 7, 14, and 28 days and studied by light and transmission electron microscopy. Macrophages and foam cells were isolated by enzymic dispersion with collagenase and cultured for further characterization by scanning electron microscopy, nonspecific esterase (NSE), and oil red O (ORO) staining. Granuloma macrophages from NC rabbits were consistently ORO-negative, contrasting with those from HC rabbits which were strongly ORO-positive, even at 4 and 7 days. With an increasing duration of exposure to hypercholesterolemia, macrophages accumulated increasing amounts of stainable lipid, and in the 28-day HC granulomas, large foam cells distended by lipid inclusions accounted for 70% of the cells present. This model has established that NSE-positive macrophages in HC granulomas accumulate lipid and assume the morphologic characteristics of atheromatous intimal foam cells.

Aortic intimal monocyte recruitment in the normo and hypercholesterolemic baboon (Papio cynocephalus). An ultrastructural study: implications in atherogenesis.

The ultrastructural features of peripheral blood monocyte margination, migration, and aortic intimal accumulation have been described in the normo- and mildly hypercholesterolemic baboon. Intimal monocyte-macrophage recruitment over fatty streaks and fibro-fatty plaques was enhanced by dietary cholesterol-fat supplementation, resulting in an 8-fold increase in monocyte-macrophages and macrophage-derived foam cells in the subendothelial space. Margination or attachment observed over both plaques and normal areas was not associated with morphologic evidence of endothelial injury. Migration through continuous aortic endothelium was principally between endothelial cells via junctions. Transitional sequences from the typical morphology of the blood monocyte to the lipid-containing macrophage or foam cell were discerned. The intimal accumulation of monocytes and macrophages reinforces our view of atherosclerosis as an inflammatory process, in which monocyte attachment is likely to reflect changes in the endothelial surface-membrane complex and surface charge, while migration to and accumulation in the SES may result from one or more chemoattractants originating in the intima or media.

Initial characterization of a peripheral blood mononuclear cell chemoattractant derived from cultured arterial smooth muscle cells.

A mononuclear cell chemoattractant of high specific activity produced by baboon (Papio cynocephalus) aortic medial smooth-muscle cells (SMCs) in culture has been partially characterized. Smooth-muscle cells, between the third and eighth passage, were grown to confluence in Medium 199 containing 10% fetal calf serum and then incubated for 24 hours in either serumless medium (Neuman and Tytell) or Medium 199 containing 0.2% bovine serum albumin. The 24-hour SMC-conditioned medium was fractionated on Sephadex G100-Superfine and potent chemoattractant activity (SMC-CF) eluted in the 10,000-12,000 dalton region. SMC-CF displayed chemotactic and chemokinetic activity for peripheral blood mononuclear cells but not for polymorphonuclear leukocytes. Production of SMC-CF by the cells was significantly inhibited in the presence of cycloheximide, and its activity was abolished after incubation with the bacterial protease subtilisin. Chromatofocusing experiments indicate that SMC-CF is a cationic protein with a pI of greater than 10.5. The role of SMC-CF may play as an inflammatory mediator in monocyte recruitment to the arterial intima in atherogenesis is discussed.

Determination of apolipoprotein A and its constitutive A-I and A-II polypeptides by separate electroimmunoassays.

Electroimmunoassays ("rocket" electrophoresis) are described for human serum apolipoprotein A and its constitutive A-I and A-II polypeptides. Purified lipoprotein A, A-I, and A-II were used to prepare monospecific antisera and to standardize assays. These specific, rapid (5-8 h), precise (the within-and between-assay coefficients of variations are 5 and 7%, respectively), and accurate (by gravimetry) assays are applicable to measurement of these polypeptides in whole serum and in various density classes of lipoproteins. Comparable results are obtained with intact and delipidized lipoproteins. Results correlated well with those obtained by radial immunodiffusion or radioimmunoassay. However, the present procedure is more rapid than the former and simpler than the latter immunoassay. Concentrations of A-I and A-II in the serum of normal men and women were similar (143 +/- 24 and 146 +/- 78 mg/dl, respectively, for A-I and 78 +/- 17 and 83 +/- 25 mg/dl for A-II). Subjects with type lla, llb, and IV hyperlipoproteinemias had similar concentrations of both polypeptides, while patients with type I disease, lecithin:cholesterol acyltransferase deficiency and LP-A deficiency had lowest concentrations of A-I (0.3-30 mg/dl) and A-II (11-20 mg/dl). The molar ratio of A-I/A-II in the serum and high-density lipoproteins was close to unity.