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The effects of long-term low-dose treatment with reserpine on plasma lipoproteins and arterial cholesterol were determined in cholesterol-fed rabbits. Hepatic low-density lipoprotein (LDL) receptors; uptake of LDL by liver, heart, and kidneys; plasma fibrinogen; blood pressure; and heart rate were also determined. Reserpine at 43 microg/kg. d was continuously infused subcutaneously via implanted minipumps for 6 weeks into conscious unrestrained male New Zealand White rabbits (n = 5) fed a 0.2% cholesterol-enriched diet. Compared with controls, reserpine (n = 4) significantly reduced the elevated levels of plasma total cholesterol and esterified and unesterified cholesterol throughout the study, and at 6 weeks of treatment these reductions were 42, 41, and 49%, respectively. The increased cholesterol in the aortic walls (n = 5) produced by the atherogenic diet was reduced by 73% (p < 0.004) and 125I-tyramine cellobiose-labeled LDL by 67 to 86% (0.05 < p <0.004), respectively. The aortic intimal-medial thickness ratio was reduced by 70%. The decrease in elevated plasma total cholesterol was mainly due to cholesterol reductions in both LDL (41%) and non-high density lipoprotein (HDL) of density < 1.019 g/ml (51%). HDL cholesterol and triglyceride levels were unchanged. Reserpine had no significant effects on the clearance of 125I-tyramine cellobiose-LDL from plasma and there was a trend towards an increase in hepatic LDL receptor expression. Heart rate was decreased by 28%. There were no significant effects on blood pressure, liver and heart lipids, hematocrit, or plasma fibrinogen. The results suggest that treatment of cholesterol-fed rabbits with reserpine at a low dose over a long period prevents increases in plasma atherogenic lipoproteins. Reserpine decreases the cholesterol in aortic walls and the intima-media thickness ratio. This anti-atherosclerotic effect of reserpine may have therapeutic implication.

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.

The effects of administering reserpine (0.1 mg/kg) or 17alpha-ethinyloestradiol (2.5 mg/kg) to New Zealand White rabbits on low density lipoprotein receptors in liver, on plasma low density lipoprotein and fibrinogen and on plasma and tissue lipids were determined. Blood pressure and heart rate were also followed. The drugs were injected subcutaneously into conscious unrestrained rabbits for 5 days. On the 6th day homologous 125I-tyramine cellobiose labelled low density lipoprotein (125I-TC-LDL) was injected intravenously and 24 h later the animals were killed. Compared to controls, reserpine significantly increased LDL receptor expression in the liver by about threefold, and reduced total cholesterol in plasma, aorta and heart, without affecting plasma triglycerides. The reductions in plasma cholesterol and heart were due to decreases in both unesterified and esterified cholesterol. Similar effects were observed with oestrogen, except that there was no change in esterified cholesterol in aorta. In liver, a decrease of 24% in total cholesterol was due mainly to decreased esterified cholesterol. In adrenal glands total cholesterol increased by 25%. Reserpine significantly accelerated the plasma clearance of intravenously injected homologous 125I-TC-LDL and reduced its accumulation in aortic wall. Neither reserpine nor oestradiol affected blood pressure, haematocrit or plasma fibrinogen. The results suggest that reserpine is an affective anti-atherogenic drug capable of decreasing cholesterol in plasma, arteries and heart by increasing high affinity LDL receptors in the liver.

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.

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.

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.

Supplementation of the diet of rabbits with fish oil or sunflower oil resulted in significant changes in the lipoproteins and lipids in serum. Compared with chow-fed rabbits, dietary fish oils decreased very low density lipoprotein (VLDL), increased low density lipoprotein (LDL), and shifted the peak of the LDL to denser fractions, whereas sunflower oil increased high density lipoprotein and shifted LDL to the lighter fractions. The amount of LDL receptors in fish oil-fed rabbit liver decreased by > 70% while there was only a small fall in these levels in sunflower oil-fed rabbit liver. The concentrations of apolipoprotein (apo) B in the subcellular organelles of the secretory compartment (rough and smooth endoplasmic reticula and Golgi fractions) were also changed by dietary lipids. In both sunflower oil- and fish oil-fed liver, apo B was increased in the lumen of the rough endoplasmic reticulum compared with fractions from chow-fed rabbit liver. The apo B in the trans-Golgi lumen from fish oil-fed livers was reduced and occurred in particles of d approximately 1.21 g/mL. In contrast, apo B in the trans-Golgi lumen from livers of sunflower oil-fed rabbits was increased and occurred in particles of d < 1.21 g/mL. These results suggests that feeding of fish oils causes an interruption in the intracellular transfer of apo B and hence assembly of VLDL. This leads to an enrichment of the rough endoplasmic reticulum membranes with cholesterol, thus downregulating the expression of the LDL receptor.

There is substantial evidence that activated components of the complement cascade are present in atherosclerotic lesions, and it was suggested some years ago that smooth muscle cells may be an important target of complement attack by the terminal components of the cascade, C5b-9, also called the membrane attack complex. Recent in vitro studies have shown that assembly of membrane attack complex on smooth muscle cells leads to the release of monocyte chemotactic protein-1, and, if this were to occur in vivo, then it could be responsible for the recruitment of monocytes into the lesion. In this study we have investigated the localization of C5b-9 in early atherosclerotic lesions of human coronary arteries, collected from autopsies, by immunohistochemical staining, C5b-9 was found to colocalize widely with smooth muscle cell alpha-actin, but not with intact macrophages, thus supporting the hypothesis that interaction of complement with smooth muscle cells may indeed be important in atherogenesis.

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.

MAC188 S/S is a monoclonal antibody which can be used in vivo to measure the absolute number of functioning low-density lipoprotein (LDL) receptors in a rabbit. The antibody binds to the extra-cellular domain of the LDL receptor and binding is not blocked by the presence of LDL. When the antibody-receptor complex is internalized, receptor recycling is inhibited for several hours. Thus when saturating doses of MAC188 S/S are administered intravenously, the amount of antibody removed from the blood (minus non-specific removal) is determined solely by the total number of LDL receptors in an animal. In this study MAC188 S/S was used to measure the number of LDL receptors in control rabbits and in animals treated with 17 alpha-ethinyl oestradiol. After treatment (which caused a 47% decrease in plasma cholesterol), receptor-mediated removal of MAC188 S/S from the blood was saturated in both groups following injection of 3.0 mg of antibody per kg body weight. Based on the amount of antibody removed via the LDL receptor at this dose, the total number of accessible LDL receptors was calculated as (2.0 +/- 0.3) x 10(15) receptors per kg body weight in control rabbits and (4.0 +/- 0.4) x 10(15) receptors per kg body weight in oestrogen-treated animals. The number of receptors in various organs was also determined. The monoclonal antibody approach therefore, allows accurate determination of LDL receptor numbers in animals with markedly different concentrations of circulating LDL, conditions in which the use of endogenous ligand would be subject to significant errors.

P388D1 macrophage-like cells have previously been shown to produce both mitogenic and inhibitory regulators of porcine smooth muscle cell (pSMC) growth. The mitogenic activity was shown to have a molecular mass of > 10 kDa while the inhibitory activity was in the range of 2-6 kDa. In the present study, we present a novel dialysis culture system where P388D1 cells were grown in dialysis membranes with a 12 kDa cut-off which allowed continuous production of fractions of the culture medium. Using pSMC as target cells, mitogenic activity was found to be retained by the dialysis membrane while the low molecular mass inhibitory activity passed freely through the membrane. The effect of the macrophage-activators phorbol myristate acetate (PMA), concanavalin A (ConA) and interferon-gamma in combination with lipopolysaccharide (IFN gamma/LPS) were investigated in the dialysis culture system. PMA, ConA and IFN gamma/LPS were found to enhance the production of mitogenic activity by P388D1 cells. PMA also increased the production of growth-inhibitory activity, while ConA abolished inhibitor production and IFN gamma/LPS had no effect on the amount of inhibitory activity produced by P388D1 cells. The experiments show that the balance of production of mitogenic and inhibitory activities by macrophages can be modulated by agents that alter the state of activation of the cells. This could be of profound significance in the influence of macrophages on smooth muscle cell growth during the development of atherosclerosis.

Murine peritoneal macrophages and the macrophage-like cell line, P388D1, were found to release both mitogenic and inhibitory modulators of growth of cells in culture. These growth factors were effective against both murine Swiss 3T3 fibroblasts and porcine aortic smooth muscle cells as assessed by [3H]thymidine incorporation into DNA and by measurement of cell number. Partial characterisation of the inhibitory activity demonstrated it to be lost on dialysis using a membrane with a 10 kDa cut-off, trypsin sensitive, heat stable, and slightly sensitive to freeze-thawing. The inhibitory activity not only affected cell growth but was found to change the morphology of porcine aortic smooth muscle cells. Gel permeation studies showed an estimated molecular mass in the range 2.5 to 6.5 kDa. The inhibitory activity could be partially purified using ion-exchange chromatography. Experiments with a neutralising antibody against transforming growth factor beta (TGF-beta) showed that TGF-beta is not responsible for the activity observed. Indomethacin had no effect on the production of inhibitor suggesting that it is not an inhibitory prostanoid. The inhibitory activity was not due to a non-specific toxic mechanism as confirmed by a [3H]adenine release assay. Incubation of P388D1 cells with cycloheximide prevented the release of inhibitory activity.

We have investigated the intracellular distribution of apolipoprotein B (apo B) in rabbit liver by immunoblotting, radioimmunoassay (r.i.a.) and enzyme-linked immunoassay (e.l.i.s.a.). Apo B100 was detected in total microsomes, rough microsomes, smooth microsomes, trans-enriched Golgi and cis-enriched Golgi and membrane and cisternal-content subfractions prepared from these fractions. There was also evidence of degradation of apo B100 in the Golgi membrane fractions. The amount of apo B in the subcellular fractions detected by competitive r.i.a. or e.l.i.s.a. ranged from 1.5 micrograms/mg of protein in the rough endoplasmic reticulum to 13 micrograms/mg of protein in the trans-Golgi fraction. Using internal standards (NADPH-cytochrome c reductase for the endoplasmic reticulum and galactosyltransferase for the Golgi membranes) it was calculated that all the apo B of liver is recovered within the secretory compartment, with 63% of the total apo B in the endoplasmic reticulum and the remainder in the Golgi. When the subcellular fractions were separated into membranes and cisternal contents, 60%, 50%, 60% and 30% of the total apo B was recovered in the membrane of the rough microsomes, smooth microsomes, cis-Golgi and trans-Golgi respectively. Using competitive e.l.i.s.a. we found that the membrane-bound form of the apo B was exposed at the cytosolic surface of the intact subcellular fractions. These observations are consistent with a model for assembly of very-low-density lipoproteins (VLDL) in which newly synthesized apo B is incorporated into a membrane-bound pool and a lumenal pool. The membrane-bound pool not used for VLDL assembly may be degraded, possibly in the Golgi region.

The effect of transforming growth factor-beta 1 (TGF-beta 1) and interleukin-1 beta (IL-1 beta) on LDL receptor in Hep G2 cells was investigated. A greater than two-fold stimulation of the binding and internalisation of [125I]-labelled LDL at 37 degrees C was observed after an 18-h incubation of the cells with TGF-beta 1 at 50 ng/ml and IL-1 beta at 11,700 units/ml compared with control cells. Scatchard analysis of the binding of [125I]-labelled LDL at 4 degrees C after an 18-h incubation of the cells with 1170 units/ml IL-1 beta and 5 ng/ml TGF-beta 1 showed that they were both acting primarily by increasing LDL receptor number. The increase in LDL receptor activity could not be attributed to an increase in cell proliferation as TGF-beta 1 at concentrations from 0.05 ng/ml to 50 ng/ml had no significant effect on either cell number or [3H]thymidine incorporation into DNA whilst IL-1 beta inhibited DNA synthesis by more than 80% at a concentration of 11,700 units/ml but had significant effect on cell number. Cholesterol biosynthesis from [14C]acetate, in contrast to the stimulation of LDL receptor activity, was inhibited by approximately two-fold by incubation with TGF-beta 1 at 50 ng/ml and IL-1 beta at 11,700 units/ml.

Paracrine factors may modulate low density lipoprotein (LDL) receptor activity in hepatocytes. To study this the effect of conditioned medium prepared from a range of cell types on the binding and internalisation of 125I-LDL in Hep G2 cells was studied. Seven of the fourteen conditioned media tested, including those from P388D1, U937, porcine smooth muscle (Pc SMC) Swiss 3T3, STO, = 48 and MDCK cells, were found to increase the binding and internalisation of 125I-LDL at 37 degrees C by Hep G2 cells (P < 0.01). The largest increase in LDL receptor activity was produced by conditioned medium from Pc SMC cells and was, therefore, selected for further analysis. The Pc SMC-conditioned medium increased LDL receptor number in Hep G2 cells by three-fold but had no effect on LDL receptor activity in human skin fibroblasts. DNA synthesis and cholesterol synthesis by Hep G2 cells were inhibited by Pc SMC-conditioned medium. Preliminary characterisation of the Pc SMC-derived factor(s) suggests that it is a protein(s) of low relative molecular mass.

A serum-free chemically defined medium (CDM) has been developed which sustains the growth in culture of the highly differentiated human hepatoma cell line Hep G2. Unlike rodent hepatoma lines, Hep G2 cells in serum-free medium have an absolute requirement for lipoprotein lipids (either low density lipoprotein (LDL) or high density lipoprotein (HDL)) for growth. In the presence of LDL (or HDL) growth was further enhanced by insulin, triiodo-L-thyronine, 17 alpha-ethinylestradiol but not by epidermal growth factor (EGF). On type I collagen gels cells cultured in CDM were contact inhibited and formed monolayers. This contrasted with the pattern of growth of cells cultured in the presence of serum on type I collagen gels and cells cultured on tissue-culture plastic in either CDM or medium containing serum which formed foci of multilayered cells. Expression of the LDL receptor and HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase genes was comparable in Hep G2 cells cultured in CDM and serum-containing medium. Furthermore, the binding and internalisation of 125I-LDL at 37 degrees C was modulated by hormones that have previously been shown to affect LDL receptor levels in liver in vivo or in hepatocytes cultured in serum-containing medium in vitro. The culture system described provides a basis for studying the regulation of hepatocyte-specific functions by soluble factors (either plasma- or cell-derived) and cell-substratum interactions in a human liver cell line.

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.

MAC188 is a rat anti-[low-density lipoprotein (LDL) receptor] monoclonal antibody (McAb) which binds to the cell surface receptor with high affinity at physiological temperatures, even in the presence of high concentrations of the natural ligand, LDL. Binding of McAb MAC188 at 37 degrees C is followed by internalization and intracellular sequestering of the receptor, which results in the transient disappearance of the receptor from the cell surface. The high binding affinity and epitope specificity of McAb MAC188 suggested that this antibody could be used to quantify receptor expression in vivo. Mixtures of radiolabelled anti-receptor antibody and a control McAb (MAC221) were injected intravenously into rabbits, and the clearance from serum and uptake into tissues was determined. A fraction of the anti-receptor McAb was cleared rapidly from the circulation by a high-affinity and saturable (receptor-dependent) process. Receptor-dependent uptake of the anti-receptor McAb was measurable in liver, adrenal glands, kidneys, spleen, kidney, thoracic aorta and heart. It was highest in liver and adrenal glands and correlated well with the level of receptor protein and the rate of LDL transport in individual tissues. Anti-receptor McAbs such as MAC188, with suitable domain specificity and binding affinity at physiological temperatures, have important advantages over the natural ligand as tracers for the receptor in vivo, and may find widespread applications in studies of the receptor status (activity) in animals and man.

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.

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.

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.

The effect of diet-induced moderate hypercholesterolemia on endothelial healing has been investigated in the rabbit following a narrow superficial injury to aortic endothelium without damage to the media of the vessel. The healing process was compared with that observed in normocholesterolemic animals. The degree of platelet involvement was similar in both normo- and hypercholesterolemic animals. Reendothelialization occurred within 48 hours in both groups of animals, showing that hypercholesterolemia did not delay endothelial healing. It was found that esterase-positive cells, which morphologically resembled monocyte-macrophages, adhered to and penetrated regenerated endothelium only in hypercholesterolemic animals. After reendothelialization in normocholesterolemic animals, there was no increase in the number of cells within the intima of the vessel and no evidence of lipid accumulation. In hypercholesterolemic animals, cells accumulated in the intima in areas of regeneration, and lipid accumulation occurred within both the intima and the media in areas of regeneration.

125I-labelled poly(vinylpyrrolidone) was used as a marker of fluid-phase pinocytosis in cultured pig arterial smooth-muscle cells. The rate of pinocytosis was temperature-dependent. A decrease in cellular ATP concentrations as a result of inhibition of either glycolysis or oxidative phosphorylation was associated with a similar decrease in pinocytosis. A microfibrillar-disruptive agent, cytochalasin B, caused a concentration-dependent stimulation of pinocytosis, whereas the microtubular-disruptive agents colchicine and vinblastine decreased pinocytosis to approximately half of control values at all concentrations used. These results indicate that fluid-phase pinocytosis in smooth-muscle cells is dependent on a continuing supply of energy and the integrity of the microtubules. Furthermore, microfilaments appear to exert a certain degree of constraint on pinocytosis, possibly by restricting invagination of the plasma membrane.

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.

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.

Aortic allografts in rabbits were observed by scanning electron microscopy 24 hr after transplantation. The extent of the leucocytic invasion could be precisely and reproducibly measured. Azathioprine and prednisolone, whether alone or in combination, given on three occasions--10 hr before, immediately after and again 10 hr after surgery, significantly inhibited the cellular invasion. Promethazine produced a slight, but not statistically significant, effect.

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.

The rationale for this study was to determine whether in the hypercholesterolemic rabbit any evidence of endothelial injury could be detected prior to or during the early phase of fatty intimal lesion formation. The data presented showed that in the first 12 weeks of feeding a 0.1% cholesterol-rich diet, rabbit aortas were covered with an intact endothelium. Focal areas of increased endothelial cell replication were observed adjacent to the aortic ostia at 12 and 20 weeks. These replicating cells were almost exclusively located at the shoulders of large raised lesions. In a similar fashion, adherent leukocytes were observed adjacent to the aortic ostia, and at later times they were concentrated at the periphery of these intimal lesions. Smooth muscle cell replication, as assessed by autoradiography, was found to be significantly increased only after 20 weeks of feeding the lipid-rich diet. These data suggest that an increased endothelial cell turnover and leukocyte adhesion were the first detectable changes induced by cholesterol feeding and that smooth muscle cell proliferation occurred soon after these events.

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.

The authors have developed an organ culture system in which segments of rabbit aorta were used for the study of the healing of superficial, intimal injury analogous to that produced in vivo by a nylon monofilament catheter. Aortic segments containing an intact endothelial monolayer were obtained for culture by careful avoidance of the damaging consequences of loss of vascular pressure, vasocontraction, and thrombosis. The segment could be maintained in culture for several weeks. Repair of superficial injury was studied after placing a series of precisely located injuries, 100 mu wide, on each aortic segment over a period of several days. The pattern of healing observed was similar to that observed in vivo. The wounded area was recovered by migrating cells within 24 hours, and during succeeding days these cells underwent a predictable pattern of remodeling which restored the normal morphologic features of the endothelium. The cells within the wound underwent a burst of cell division between 24 and 96 hours after injury and subsequently became quiescent. This organ culture approach potentially allows the investigation of many consequences of endothelial injury in a system which provides far greater control and manipulation of the cellular environment than is possible in vivo.

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.

Interactions in vivo between C-reactive protein (CRP) and apolipoprotein B (apo-B)-containing lipoproteins were sought in inflammatory lesions and atherosclerosis. CRP was demonstrated immunohistochemically on the surface of some muscle fibres in locally induced inflammatory lesions in the rabbit, but apoB was not detected in the same distribution. CRP was not detected in catheter-induced aortic endothelial injuries in the rabbit, in arterial lesions containing apoB from cholesterol-fed rabbits, in apoB-containing human fatty streaks or in advanced human atherosclerotic lesions.

A modification of the semiautomated assay method of Antonis (1965. J. Lipid Res. 6:307-312) for free fatty acid is presented. Free fatty acids are extracted from serum or plasma into di-n-butyl ether-2-methoxyethanol; the extract is almost free from phospholipids. The acids are analyzed in a portion of extract by a copper soap method using diphenylcarbazide. The extractant, being less dense than water, is easily separated from an aqueous phase both in the extraction of samples and in the assay of copper soaps. The assay is comparable in accuracy with well-tried titrimetric methods and is quicker and easier to operate.

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.

Intradermal injection of rabbits with lipid-coupled bovine serum proteins or bovine serum itself prior to a regimen of intravenous injections of bovine serum enhanced the incidence of joint lesions over that seen in animals receiving the intravenous regimen only. This increased of synovial lesions appeared, however, to be unrelated, either to the state of delayed hypersensitivity or to the concentration of passive haemagglutinating antibodies to bovine serum proteins in these animals. Lipid-coupled bovine serum, given as a first course in a regimen of intravenous injections, was no more arthritogenic than the native proteins and, as a second course series of intravenous injections, was definitely less arthritogenic.

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.

Electron microscopic analysis of 500 negatively stained coated vesicles isolated from human placenta showed that they exist within limits in a continuous range of volumes with an unimodal distribution. Some vesicles were larger than the frequently quoted maximum size of these organelles (diameter 100nm). The ratio of hexagonal to pentagonal facets in the clathrin lattice of the vesicle wall appears to be variable. This feature may be important in morphogenesis since the mean volume of prolate vesicles is larger than that of spherical vesicles. Empty lattices had a mean volume smaller than that of lattices containing phospholipid bilayers.

A BASIC program is described for acquisition of data and data reduction for an automated densitometric system for quantitation of lipids separated by high-performance thin-layer chromatography. The program allows calculation of mass of samples from log/log calibration curves computed from standards. The calculated masses are reported as nmol/volume or nmol/mg protein. The program contains a flexible dialog system which permits its use for a variety of applications in addition to the system described for quantitation of lipids.

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.

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.