David E. Bowyer

C-Reactive Protein Frequently Colocalizes With the Terminal Complement Complex in the Intima of Early Atherosclerotic Lesions of Human Coronary Arteries

There is increasing evidence that complement activation may play a role in atherogenesis. Complement proteins have been demonstrated to be present in early atherosclerotic lesions of animals and humans, and cholesterol-induced atherosclerotic lesion formation is reduced in complement-deficient animals. Potential complement activators in atherosclerotic lesions are now a subject matter of debate. C-reactive protein (CRP) is an acute-phase protein that is involved in inflammatory processes in numerous ways. It binds to lipoproteins and activates the complement system via the classic pathway. In this study we have investigated early atherosclerotic lesions of human coronary arteries by means of immunohistochemical staining. We demonstrate here that CRP deposits in the arterial wall in early atherosclerotic lesions with 2 predominant manifestations. First, there is a diffuse rather than a focal deposition in the deep fibroelastic layer and in the fibromuscular layer of the intima adjacent to the media. In this location, CRP frequently colocalizes with the terminal complement complex. Second, the majority of foam cells below the endothelium show positive staining for CRP. In this location, no colocalization with the terminal complement proteins can be observed. Our data suggest that CRP may promote atherosclerotic lesion formation by activating the complement system and being involved in foam cell formation.

Inhibitory effect of calcium antagonists on balloon catheter-induced arterial smooth muscle cell proliferation and lesion size

Calcium antagonists inhibit atherogenesis in the cholesterol-fed rabbit without producing hypolipidaemia, suggesting a direct action on the arterial wall. In this study, the effects of several calcium antagonists on the myoproliferative response to balloon catheter injury of the aorta have been investigated in normolipidaemic rats and rabbits. The incorporation of [3H]thymidine into rat aortic DNA 48 h after balloon injury was markedly reduced by twice daily oral administration of nifedipine, verapamil, diltiazem or lanthanum. DNA synthesis in other proliferating tissues was unaffected. Twice daily oral administration of prazosin or minoxidil, antihypertensive agents that are not calcium antagonists, also selectively reduced arterial DNA synthesis. In balloon catheterised rabbits twice daily oral administration of nifedipine (10 mg/kg) caused a 39% reduction in the cross-sectional area of the neo-intima 14 days after injury. These results show that nifedipine and other antihypertensive agents inhibit smooth muscle cell proliferation.

Processes in atherogenesis: complement activation

The complement system consists of a complex group of plasma proteins, which, on activation, lead to a cascade of interactions culminating in the production of a variety of pro-inflammatory molecules. The system also contains cellular receptors for complement fragments produced during activation and regulatory molecules. It is part of the innate immune system representing humoral defence, but in certain circumstances may itself contribute to disease. In the formation of atherosclerotic lesions, there are two outstanding cellular phenomena, monocyte recruitment, with subsequent development of lipid-filled foam cells and smooth muscle cell activation. Subendothelial deposition of low density lipoprotein appears to be an important stimulus in these events and substantial evidence suggests that complement activation may be a link between lipoprotein deposition and subsequent lesion development.

Atherosclerosis induced in hypercholesterolaemic baboons by immunological injury; and the effects of intravenous polyunsaturated phosphatidyl choline

Abstract Groups of 5–8 baboons were fed either a control or hypercholesterolaemic diet for 6 months. During the last 90 days, each group was given 5 i.v. injections of bovine serum albumin (BSA) at 16 day intervals or control injections of saline. Only those animals which were both hypercholesterolaemic and injected with BSA developed aortic and coronary atherosclerosis. An intravenous injection of 1 g polyunsaturated soya phosphatidyl choline (Lipostabil) thrice weekly into animals receiving the atherogenic diet and BSA, reduced the incidence and severity of aortic atherosclerosis but had no effect on plasma cholesterol, phospholipids or the fatty acid composition of the cholesterol esters and lecithin. Compared with controls, animals given the hypercholesterolaemic diet had increased aortic lipase and normal cholesterol esterase activity. Those given the same diet and Lipostabil had a normal aortic lipase and over 50% increase in cholesterol esterase activity. It is concluded that immunological injury hastens the onset of atherosclerosis produced by feeding a hypercholesterolaemic diet and that changes in aortic lipolytic enzymes may be the mechanism by which Lipostabil reduces atherosclerosis.

Complement-Induced Release of Monocyte Chemotactic Protein-1 From Human Smooth Muscle Cells A Possible Initiating Event in Atherosclerotic Lesion Formation

Increasing evidence suggests that complement activation might represent an important mechanism in early atherogenesis. Thus, complement components, in particular the membrane attack complex (MAC) C5b-9(m), have been isolated from human atherosclerotic lesions. Furthermore, complement activation is known to occur in atherosclerotic lesions induced in experimental animals, and the severity of cholesterol-induced plaques is markedly reduced in complement-deficient animals. During atherogenesis monocytes are recruited into the arterial wall, and a potent chemoattractant for monocytes, monocyte chemotactic protein-1 (MCP-1), is expressed by vascular smooth muscle cells (SMCs). We hypothesized that generation of MACs on SMCs during the activation of complement might lead to the release of MCP-1 and hence to monocyte recruitment. In this study, MACs were generated on human SMCs in vitro by sequential addition of the purified complement components C5b6, C7, C8, and C9. This supernatant of the culture was chemotactic for freshly isolated peripheral blood monocytes in a modified Boyden chamber. The chemotactic activity of the supernatant was abolished by anti-MCP-1 blocking antibodies but not by an isotype-matched antibody against an irrelevant antigen. The release of chemotactic activity was dependent on the dose of MAC formed on SMCs and was demonstrated within 10 minutes of exposure of the cells. The data support the hypothesis that complement-mediated release of MCP-1 from SMCs might be important in the recruitment of monocytes into the developing atherosclerotic lesion and could be an important initiating event in atherogenesis.

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.

A quantitative densitometric method for the rapid separation and quantitation of the major tissue and lipoprotein lipids by high-performance thin-layer chromatography : I. Sample preparation, chromatography, and densitometry

A rapid method for the separation and quantitation of the major lipids of tissues and lipoproteins by automated high-performance thin-layer chromatography is presented. Solvent systems for one-dimensional separation of neutral lipids, of cholesteryl esters, and of phospholipids are described. Separated lipids are measured following treatment with methanolic sulphuric acid containing manganese chloride and scanned in fluorescence or absorption mode. Absolute quantitation is obtained by the use of an internal standard and by references to standards for each lipid run on the same plates as samples. The method described here is particularly suitable for the rapid quantitation of small amounts of lipid (0.01-0.02 nmol per sample), for example in tissue culture studies; 100 micrograms of fibroblast or macrophage protein are sufficient for complete lipid analysis. The coefficients of variation due to the sample preparation, application to the plates and densitometry are in the range 7.2-9.1%. The method was compared with enzymatic determinations for cholesterol and gave correlation coefficients of 0.95 for total cholesterol and 0.91 for unesterified cholesterol. Phospholipid estimation was compared with large-plate thin-layer chromatography and phosphorus analysis and gave correlation coefficients of 0.90 for phosphatidylcholine and 0.89 for sphingomyelin.

Aortic endothelial cell morphology observed in situ by scanning electron microscopy during atherogenesis in the rabbit

The morphology of endothelial cells during the induction of atherosclerosis in the descending aortic arch of the hypercholesterol rabbit was studied in situ by scanning electron microscopy (SEM) following silver staining, fixation at physiological pressure, and air-drying of specimens- The earliest deviations from normal endothelial morphology were observed 3 weeks after starting to feed a semi-synthetic diet containing 20% beef fat and 0.2% cholesterol. These were (1) the occurrence of brightly silver stained (argyrophilic) cells, (2) areas of irregularly shaped cells which were often larger and more weakly stained than normal cells and (3) increased incidence of stigmata and stomata associated with the irregular cells. After 6 weeks of hypercholesterolaemia, similar changes were present in the endothelium, but were often also associated with sub-endothelial swelling. These represented the first atherosclerotic lesions. Following 12, 20 and 24 weeks of hypercholesterolaemia, larger raised macroscopic lesions were observed which were always endothelialized. Endothelial morphology and lesion topography suggested that early fatty streaks were composed of numerous focal swellings. In addition to the abnormal endothelial morphology noted at 6 weeks, endothelial cells overlying more advanced lesions became rounded in outline.

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.

Methods for the rapid separation and estimation of the major lipids of arteries and other tissues by thin-layer chromatography on small plates followed by microchemical assays

Methods are described for the rapid separation of the major individual phospholipids and neutral lipids of tissues by thin-layer chromatography on small glass plates (75 X 75 mm), and for the specific microchemical estimation of separated lipids and for determination of fatty acid composition and radioactivity. The overall method, involving tissues extraction, thin-layer chromatographic separation and assay has been evaluated using pure standards and biological samples and gives good reproducibility and almost complete recovery of lipids.