Rabbit and rat C-reactive proteins bind apolipoprotein B-containing lipoproteins.

Immobilized rabbit and rat C-reactive protein (CRP) were found to selectively bind apolipoprotein B (apoB)-containing lipoproteins (low density lipoprotein, LDL and very low density lipoprotein, VLDL) from whole serum in a manner similar to that previously reported with human CRP. In acute phase human serum the CRP is in a free form, not complexed with lipoprotein or any other macromolecular ligand, and in acute phase serum from most rabbits fed on a normal diet the rabbit CRP was also free. However, in acute phase serum or heparinized plasma from hypercholesterolemic rabbits part or all of the CRP was found by gel filtration and immunoelectrophoretic techniques to be complexed with beta-VLDL, an abnormal apoB-containing plasma lipoprotein present in these animals. The presence of extent in different serum samples of CRP complexed with lipoprotein correlated closely with the serum apoB concentration. The formation of complexes between native, unaggregated rabbit CRP in solution and apoB-containing lipoproteins was readily demonstrable experimentally both with the isolated proteins and in whole serum. In all cases these interactions were calcium-dependent and inhibitable by free phosphoryl choline. The present findings extend earlier work in man and the rabbit and indicate that among the C-reactive proteins from different species, which are structurally highly conserved, the capacity for selective binding to apoB-containing plasma lipoproteins is also a constant feature. These interactions may therefore be related to the in vivo function of CRP in all species and this function may in turn be relevant to pathological conditions, such as atherosclerosis, in which lipoproteins are important.

A rare case of unilateral hemifacial spasm and facial palsy associated with an abnormal anatomical variant of the posterior basilar circulation.

Tortuous vertebral arteries are a rare anatomical variant. Mild tortuosity is usually asymptomatic whereas severe tortuosity may present with ischaemic symptoms or compressive symptoms (focal neurological deficit). While a resulting hemifacial spasm has been previously described, sparse literature exists for its association with facial palsy. We present a rare case of facial spasm along with facial palsy in a 67-year-old woman who was found to have an anatomical variant in the posterior basilar circulation with an ectatic basilar artery and significantly displaced posterior vertebral artery impinging on the facial nerve.

The developmental changes in plasma adrenal androgens during infancy and adrenarche are associated with changing activities of adrenal microsomal 17-hydroxylase and 17,20-desmolase.

The plasma concentrations of dehydroepiandrosterone, androstenedione, and dehydroepiandrosterone sulfate decrease during the first year of life, remain low during childhood, and then increase during adrenarche. To determine whether alterations in adrenal enzyme activity might explain the changing secretory pattern of the adrenal androgens, we measured human adrenal microsomal 3 beta-hydroxysteroid dehydrogenase-isomerase, 17,20-desmolase, 17-hydroxylase, and 21-hydroxylase activities. 12 adrenals from individuals aged 3 mo to 60 yr were studied. The patients were divided into three groups based upon the age of the patient when the adrenal glands were obtained: group 1, infants aged 3--8 mo (n = 3); group 2, preadrenarchal or early adrenarchal children aged 2--9 yr (n = 4); and group 3, adults aged 20--60 yr (n = 5). The mean activity of the 17,20-desmolase, 17-hydroxylase, and 21-hydroxylase fell by 50% and that of 3 beta-hydroxysteroid dehydrogenase-isomerase activity rose 80% from group 1 to 2. A fourfold increase in 17,20-desmolase (P less than 0.002) and 17-hydroxylase (P less than 0.001) activity and a doubling in 21-hydroxylase activity (P less than 0.005) occurred between groups 2 and 3. We conclude that the decline in plasma adrenal androgens after birth appears to be associated with a rise in 3 beta-hydroxysteroid dehydrogenase-isomerase and a fall in 17,20-desmolase and 17-hydroxylase activity. The subsequent increase in plasma adrenal androgen concentration during adrenarche is coincident with a rise in 17,20-desmolase and 17-hydroxylase activity.

Rabbit small intestinal brush border membrane preparation and lipid composition.

1. Rabbit intestinal brush border membrane vesicles were prepared either from frozen or fresh tissue and the lipid composition was analysed. 2. Lipids extracted from membranes prepared by the Ca2+-precipitation method (Schmitz, J., Preiser, H., Maestracci, D., Ghosh, B.K., Cerda, J.J. and Crane, R.K. (1973) Biochim. Biophys. Acta 323, 98--112; Kessler, M., Acuto, O., Storelli, C., Murer, H., Müller, M. and Semenza, G. (1978) Biochim. Biophys. Acta 506, 136--154) had exceptionally high levels of lysophospholipids and free fatty acids. An intrinsic, Ca2+-activated phospholipase A is responsible for the lipid decomposition. 3. It was necessary to modify the preparation of brush border membranes. Essentially, EGTA is used to keep the free Ca2+ concentration low so that phospholipases are inactivated, and Mg2+ instead of Ca2+ is employed to aggregate selectively contaminating membranes. The modified procedure gives membranes suitable for lipid analysis. 4. The molar ratio of neutral, phospholipid and glycolipid is about 1 : 1 : 1. The major neutral lipids are free cholesterol and fatty acids. The major phospholipids are phosphocholine-containing (approx. 45%) and phosphatidyl-ethanolamine (approx. 40%). The remainder (15--20%) is made up ppf acidic phospholipids, mainly phosphatidylserine and phosphatidylinositol. The major glycolipid is ceramide monohexodise. 5. The major fatty acids of total lipids and phospholipids are palmitic, stearic, oleic and linoleic acids. Individual phospholipids are characterised by distinct fatty acid compositions and differ markedly in the ratio of unsaturated-to-saturated fatty acid. X

Human blood-derived macrophages induce apoptosis in human plaque-derived vascular smooth muscle cells by Fas-ligand/Fas interactions.

Human atherosclerotic plaques that rupture are characterized by relatively low vascular smooth muscle cell (VSMC) and high inflammatory cell contents. Ruptured plaques also contain higher numbers of apoptotic VSMCs than do stable lesions, suggesting that VSMC apoptosis may promote plaque rupture. We examined the ability of human monocytes/macrophages to induce apoptosis of VSMCs derived from human carotid plaque, aortic media, and coronary media. Macrophages, but not T lymphocytes, induced a dose-dependent apoptosis of VSMCs, which required monocyte maturation to macrophages and direct cell-cell contact/proximity. VSMC apoptosis was inhibited by neutralizing antibodies to Fas-ligand (Fas-L) or an Fas-Fc fusion protein, indicating the requirement for membrane-bound Fas and Fas-L. Monocyte maturation was associated with increased surface expression of Fas-L, coincident with the onset of cytotoxicity. VSMCs expressed surface Fas, which was increased in plaque VSMCs, and plaque VSMCs also underwent Fas-induced apoptosis. We conclude that human macrophages potently induce human VSMC apoptosis, which requires direct cell-cell interactions and is in part dependent on Fas/Fas-L interactions. Macrophage-induced VSMC apoptosis may therefore directly promote plaque rupture.

Membrane-bound apolipoprotein B is exposed at the cytosolic surface of liver microsomes.

We have used a competitive enzyme-linked immunoassay with a panel of monoclonal antibodies to probe the topography of the membrane-bound form of apolipoprotein B (apo B) in rabbit microsomes. All epitopes investigated were found to be expressed at the cytosolic side of the microsomal membrane under conditions in which the vesicles remained sealed. These results indicate that the membrane-associated form of apolipoprotein B is either at the cytosolic side of the endoplasmic reticulum membrane or integrated into the membrane. From this site apo B may be translocated to the lumen for assembly into VLDL or may be degraded.

Effect of concentration of perfusing free fatty acid on arterial lipid synthesis in perfused normal and atherosclerotic rabbit aortas.

Normal and atherosclerotic rabbit aortas were perfused at physiological pressure for 1 hour with media containing various concentrations of [3H]oleic acid, between 0.5 and 2.0 mmoles/l, complexed to a fixed concentration 40 g/l of bovine serum albumin (BSA). The mass of free fatty acid (FFA), which entered the arterial wall and was subsequently utilised for lipid synthesis, was calculated from the measured specific activities of FFA in the perfusates. In normal tissue, at all concentrations of FFA in the perfusate, the highest rates of utilisation of perfusate FFA for arterial lipid synthesis were for phospholipids (PL) and triglycerides (TG), with only about 2% in cholesteryl esters (CE). In atherosclerotic tissue, at both low and high concentrations of perfusate FFA, about 25% of fatty acid entering arterial lipids was in CE. When the concentration of FFA in the perfusion medium was raised, the mass of FFA from the medium that was incorporated in the total arterial lipids, increased in both normal and atherosclerotic tissue. The increase was due in normal tissue, to significant increases in incorporation into FFA, lecithin (PC), phosphatidyl inositol (PI), phosphatidyl ethanolamine (PE), TG and CE, whilst in atherosclerotic tissue it was due to increased incorporation into PC, PI, TG and CE. The results suggest that raised concentrations of FFA in blood may increase the rate of synthesis of lipids in normal and atherosclerotic tissue and thus exacerbate the accumulation of certain lipids such as cholesteryl esters, in fatty streak lesions of atherosclerosis.

Scanning electron microscopy: arterial endothelial integrity after fixation at physiological pressure.

The luminal surface of normal rabbit aorta was examined by scanning electron microscopy (SEM) after outlining endothelial cells by staining intercellular junctions with silver. When aortas were fixed in situ at physiological pressure before processing for SEM, a reliable assessment of the morphological integrity of the endothelium was possible. In contrast, when aortas were excised and placed in fixative, contraction of the sub-endothelial structures made interpretation of endothelial integrity difficult.

Distortion of endothelial repair. The effect of hypercholesterolaemia on regeneration of aortic endothelium following injury by endotoxin. A scanning electron microscope study.

Five young male New Zealand White rabbits were fed a semi-synthetic diet containing 0.2% cholesterol for 2 weeks and a control group of 5 animals was fed a normal stock diet. All animals were then injected intravenously with a single dose of endotoxin from Serratia marcescens (200 microgram/kg body weight) and continued on their respective diets for a further 4 weeks. The aortas were then stained with silver nitrate and fixed under pressure for Scanning Electron Microscopy (SEM). Argyrophilic endothelial cells were present in both groups of animals 4 weeks after endotoxin injections. In the cholesterol-fed animals, however, these cells were often covered with pits and craters. These findings suggest that the hypercholesterolaemia may affect the regeneration of arterial endothelial cells.

Scanning electron microscopy: morphology of aortic endothelium following injury by endotoxin and during subsequent repair.

A single injection of endotoxin P45 Poly Serratia marcescens was used to induce endothelial injury in rabbits. The aortic endothelium was examined by Scanning Electron Microscopy (SEM), at various times after administration of endotoxin, using the technique of silver staining and pressure fixation. Within one hour after injection, some endothelial cells were curled-up and spindle-shaped in appearance. Areas of aorta devoid of endothelial cover were occasionally observed and platelets were sometimes found adhering to these sites. Two and four weeks after initial injury no spindle-shaped cells were found. Instead, some endothelial cells were heavily stained with silver. Small denuded zones were still found and these were surrounded by brightly silver-stained cells. This study confirms that endotoxin rapidly causes endothelial injury and suggests that regenerating endothelial cells which were formed following injury are avidly stained by silver salts and appear as bright cells by SEM.

Scanning electron microscopy of arteries. The morphology of aortic endothelium in haemodynamically stressed areas associated with branches.

The endothelial surface around branches of the normal rabbit aorta was examined by scanning electron microscopy (SEM). Focal endothelial damage was consistently found both on, and distal to, aortic flow dividers. In some case small areas were denuded of endothelial cover. Similar injury to endothelal cells was also occasionally observed proximal to the ostium of a branch. Haemodynamic forces, expecially high shear stress, may be responsible for these morphological changes. This altered endothelial integrity probably underlies the susceptibility of these sites to the formation of induced atherosclerotic lesions.

Macrophages modify beta-VLDL by proteolysis and enhance subsequent lipid accumulation in arterial smooth muscle cells.

Murine resident peritoneal macrophages (MRPM), incubated with beta-very low density lipoprotein (beta-VLDL), modify the beta-VLDL, producing an increase in the mobility of the lipoprotein. The modification does not result in an increase of thiobarbituric acid-reactive substances (TBARS) in the lipoprotein, and is not inhibited by butylated hydroxyanisole (BHA), EDTA, removal of copper and iron from the medium, or by diphenyliodonium (DPI), suggesting that the mechanism of modification is independent of oxidation. Macrophage conditioned medium performed the modification in the absence of cells, and phenylmethylsulphonyl fluoride (PMSF) inhibited beta-VLDL modification, whereas other protease inhibitors did not, suggesting that a secreted neutral serine protease may possibly be involved in the mechanism. The modified beta-VLDL enhanced the accumulation of cholesterol esters by smooth muscle cells (SMC).

Inhibition of pinocytosis and induction of release of lysosomal contents by lysosomal overload of arterial smooth muscle cells in vitro.

Lysosomal overload was induced experimentally in cultured aortic smooth muscle cells by incubation with chloroquine or sucrose. Lysosomal overload was accompanied by a marked reduction in pinocytosis and induced the release of lysosomal contents into the medium. Thus, previously accumulated pinocytic tracer and beta-glucuronidase, a lysosomal enzyme, were released into the medium whereas lactate dehydrogenase, a cytosolic enzyme, was not.

Monocyte prostaglandins inhibit procollagen secretion by human vascular smooth muscle cells: implications for plaque stability.

Extracellular matrix remodelling occurs during atherosclerosis dictating the structure of the plaque and thus the resistance to rupture. Monocytes and macrophages are believed to play a role in this remodelling. In the present study, filter-separated co-culture has been used to study the effect of monocytes on procollagen turnover by human vascular smooth muscle cells (VSMC). In this system, freshly isolated human peripheral blood monocytes inhibited procollagen secretion from VSMC without affecting either degradation of procollagen, or DNA synthesis by the VSMC. Insertion of a 12 kDa dialysis membrane between the two cell types and treatment with indomethacin showed that the inhibitory factor was of low molecular weight and was cyclooxygenase-dependent. Pre-incubation of each cell type with indomethacin demonstrated that monocyte, but not VSMC cyclooxygenase was required. Thus, the inhibitory effect on procollagen secretion was due, most likely, to monocyte prostaglandins. Neither inhibition of thromboxane synthetase, nor blocking IL-1 activity, reduced the inhibitory activity. Addition of prostaglandins PGE1, PGE2 and PGF2alpha to VSMC cultures caused a reduction in procollagen secretion which was equivalent to, but was not additive with, the maximal effect achieved by monocytes. Monocytes and macrophages are a major source of prostaglandins and these molecules are likely to play an important role in collagen turnover within lesions.

A standardised method of culturing aortic explants, suitable for the study of factors affecting the phenotypic modulation, migration and proliferation of aortic smooth muscle cells.

The study of factors affecting phenotypic change and growth of aortic smooth muscle cells (SMC) typically involves either the isolation of SMC by enzymatic dissociation or observation of outgrowth of cells from primary explants of vascular tissue. Explants provide a system in which the growth of cells can be investigated without dissociating them totally from their normal environment and avoids some of the problems of variability associated with enzymatic digestion. We describe here a standardised method for the preparation of medial explants of arterial tissue using a McIlwain tissue chopper, which is both fast and reproducible. Measurement was made of the percentage of explants showing outgrowth and of the distance migrated by cells at various times after plating explants singly into wells of a 96-well plate. Using this method, by 12 days after explanting, more than 95% of explants from normal rabbit aorta had shown outgrowth, in contrast to only 50% of explants prepared using a scalpel blade. Explants from atherosclerotic rabbit aorta showed a shorter lag phase before outgrowth commenced than explants from normal rabbit aorta of a similar age, but the subsequent rate of growth was the same. In contrast, when explants of normal rabbit aorta were grown in hyperlipidic rabbit serum, the lag phase was the same as for normal serum, but the subsequent rate of growth was greater. Explants from normal rabbit aorta treated with heparin showed an increased lag phase but reduced rate of growth. Treatment with heparinase decreased the lag phase and increased the rate of growth as did elastase.

A sensitive RNase protection assay for the quantitation of the mRNAs for the LDL receptor and HMG-CoA reductase in human total RNA. Effects of treatments on cells in culture designed to up- and down-regulate expression of the LDL receptor.

Regulation of expression of the genes for the low density lipoprotein receptor (LDLR) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is of central importance in the control of cholesterol metabolism and thus in influencing the concentration of low density lipoprotein in the plasma. This can be studied by investigating the effects of factors (hormones, drugs, etc.) on the levels of mRNA for these genes. An RNase protection assay is reported for measurement of the levels of mRNA for the LDLR and HMGR. Several probes have been developed for these genes, together with probes for the "housekeeping" genes, beta-actin and glyceraldehyde-3-phosphate dehydrogenase. Various conditions in the assay have been examined and optimised, e.g. conditions for solution hybridization and RNase digestion and the use of "sense" RNA standards. The assay allows accurate measurement of approximately 2 x 10(7) copies of LDLR and HMGR mRNAs, which is equivalent to the number of copies present in approximately 1 x 10(6) human dermal fibroblasts and approximately 5 x 10(5) Hep G2 liver hepatoma cells cultured in 10% fetal calf serum. The average number of copies of mRNA per cell was estimated in fibroblasts and Hep G2 cells under various conditions of regulation of the LDLR and revealed the following: [table: see text] Under the chosen conditions 10 copies per cell was the detection limit for the assay. The effect of these treatments on the number of copies of mRNA per cell for beta-actin and glyceraldehyde-3-phosphate dehydrogenase was also determined.

Narrow superficial injury to rabbit aortic endothelium. The healing process as observed by scanning electron microscopy.

A study was made of the healing of aortic endothelium in rabbits following the production of a defined superficial injury. This was induced using a fine nylon filament which removed the endothelial cells without producing significant damage to underlying structures. The morphology of the injury and subsequent repair was observed using light microscopy and scanning and transmission electron microscopy. Two forms of injury were produced (a) a longitudinal injury along the full length of the aorta which was 50-80 microns wide (about 5-8 cell widths), (b) a circumferential injury approximately 80 microns wide (about 2 cell lengths). Thirty minutes after injury the exposed tissue was almost devoid of adherent cells, but after 4 h became covered by a sparse monolayer of platelets. Occasional leukocytes were also present from 7 h after injury. Injury tracks were found to repair very quickly; re-endothelialisation being complete by 48 h and there being no sign of injury by 7 days.

Inhibition of human arterial smooth muscle cell growth by human monocyte/macrophages: a co-culture study.

Monocyte/macrophages produce a variety of substances which may influence the function of smooth muscle cells (SMC). During atherogenesis, macrophages are thought to modulate SMC migration, proliferation and synthesis of extracellular matrix. Such modulation is the balance between stimulatory and inhibitory influences. Thus, for example, our earlier studies have shown that macrophages not only secrete mitogens, but also produce small molecular weight inhibitors of SMC proliferation. In the present study, we have used a co-culture system in which human monocyte/macrophages were separated from human arterial SMC (hSMC) by a filter with the optional addition of a 12 kDa cut-off dialysis membrane, in order to assess their effect on hSMC growth. We have found that human peripheral blood-derived monocytes produced a substance of < 12 kDa that inhibited hSMC growth in the co-culture system. The monocyte-derived factor causing this effect was completely blocked by indomethacin, indicating that growth-inhibitory factors produced by the monocytes were cyclooxygenase products. We have shown that PGE1 and PGE2 inhibit hSMC growth, making them likely candidates for the effector molecules released from monocytes in our co-culture system.

Endothelial healing following defined injury to rabbit aorta. Depth of injury and mode of repair.

A study has been made of the healing of a narrow deep injury to rabbit aortic endothelium which also involves damage to the media of the vessel. The injury was produced using a nylon catheter containing a wire filament; the injury was approximately 150 micron in width and damaged up to 3 elastic lamellae. Immediately after injury platelet aggregates were observed over the injured areas, several hours later large numbers of leukocytes were also seen to adhere. Two days after injury a non-thrombogenic neointimal surface was observed over deeply injured areas; endothelial cells could be identified covering the injured area at 6 days. The healing process following the injury has been directly compared with the healing of rabbit aortic endothelium following a superficial injury of similar width, where endothelial cells are removed without significant damage to the media of the vessel [1]. The results show that (a) following a narrow injury to the aorta which causes damage to the media platelet aggregation and proliferation of smooth muscle cells occurs, (b) despite the disruption of subendothelial components, endothelium rapidly regenerates over the narrow injured area, although not as quickly as for a superficial injury.

Connective tissue responses to oxysterols.

We studied the effect of sterols and oxidized sterols on promoting tissue inflammation and necrosis, and on causing cell death in tissue culture. The cells studied were mouse fibroblasts and macrophages, and pig vascular smooth muscle. The lipid preparations were dibromide-purified cholesterol, air-oxidized cholesterol, 25-hydroxycholesterol, cholesterol-5alpha, 6 beta-epoxide and cholestane 3 beta, 5 alpha, 6 beta-triol. Of these, the triol was most active in its cytopathic effect on cells in culture, in size of granuloma formation and in producing necrosis. The epoxide, 25-hydroxycholesterol and air-oxidized cholesterol produced rather less effect on tissues and cultures than the triol, but more than purified cholesterol and the control. The results are considered in relation to the presence of oxysterols in stored foodstuffs and their possible presence over the surface of cholesterol crystals in the tissues.