Tom Björnheden

Accumulation of lipoprotein fractions and subfractions in the arterial wall, determined in an in vitro perfusion system

A large proportion of a dense subfraction of LDL in plasma is coupled with an increased risk of coronary artery disease, CAD. This may reflect an increased inflow of such LDL subfractions into the intima, since the inflow of lipoproteins is supposed to be inversely related to the size of the particles. In order to evaluate this possibility we used an in vitro perfusion system for aortic intima-media from rabbits with experimental atherosclerosis. The uptake of human VLDL, LDL, HDL and subfractions of LDL (LDL1, 1.019-1.035 and LDL2, 1.035-1.063 g/ml) in lesions and non-involved areas was studied. Our results indicate that particle size is an important factor for the clearance of lipoproteins into the arterial tissue, both for plaques (VLDL 7.6, LDL 25, HDL 58 nl/mg wet wt./h) and in other areas (VLDL 3.8, LDL 4.1, HDL 12 nl/mg wet wt./h). Interestingly, the uptake of LDL2 was as much as 1.5-1.9 times higher than LDL1. This supports the view that an increased lipid load in the arterial wall may be one mechanism behind the association between denser LDL and CAD. Our data also suggest that the difference between LDL uptake in plaque (576 nl/mg wet wt.) and other areas (48 nl/mg wet wt.) not only reflects a rapid clearance but a large distribution volume of the intima (plaque > 60%, non-involved areas 5.7%).

Hypoxia increases 25-hydroxycholesterol-induced interleukin-8 protein secretion in human macrophages

Interleukin-8 (IL-8) is a chemotactic factor for T-lymphocytes and smooth muscle cells and may therefore have an important effect in atherogenesis. It is secreted from oxysterol-containing foam cells which have been found in hypoxic zones in atherosclerotic plaques. The aim of this study was to investigate the effect of hypoxia on the secretion of IL-8 by oxysterol-stimulated macrophages. Hypoxia enhances 25-hydroxycholesterol (25-OH-chol)-induced IL-8 secretion in human monocyte-derived macrophages. The effect is most pronounced when macrophages are incubated with low concentrations of 25-OH-chol. Both 25-OH-chol and hypoxia increases the intracellular level of the signalling molecule hydrogen peroxide (H(2)O(2)). This event coincided with an enhanced binding of the transcription factor c-jun to the IL-8 gene promoter and an increased IL-8 mRNA expression in hypoxic macrophages. These observations suggest that similar intracellular signalling pathways are used for both 25-OH-chol-induced IL-8 expression and hypoxia-induced IL-8 expression. Thus, hypoxia in atherosclerotic plaques may increase the secretion of IL-8 from oxysterol-containing foam cells, which subsequently may accelerate the progression of atherosclerosis.

Fc-dependent binding of monocytes to areas with endothelial injury in the rabbit aorta

Abstract IgG accumulates in injured endothelial cells and nonendothelialized intima. In experimental atherosclerosis, such areas contain large amounts of monocytes. The hypothesis was raised that IgG in areas with endothelial injury (defective endothelium) stimulates monocyte binding via the Fc receptor. This hypothesis was tested in an in vitro system using segments of rabbit thoracic aorta in perfusion chambers. The chambers were initially perfused with autologous serum, permitting the binding of IgG to defective endothelium. Then, autologous mononuclear cells were injected. Monocytes adhered preferentially to defective endothelium, as indicated by a 10-fold increase when compared to intact endothelium. Some lymphocytes also adhered to defective endothelium, but there was a 4-fold increase in the monocyte/lymphocyte ratio on defective endothelium, as compared to the injected cells. The number of monocytes on defective endothelium was reduced by 40% if the aortas were perfused with Staphylococcal Protein A, an Fc-binding protein, prior to injection of cells. No effect of Protein A on lymphocyte adhesion could be detected. An equally large reduction of monocyte adhesion was observed in segments initially perfused with IgG-depleted serum instead of native serum. Scanning and transmission electron microscopy indicated that monoctes adhered to intimal fibers, and also to injured endothelial cells. These data indicate that monocytes selectively adhere to defective endothelium, and that the adhesion is in part mediated via the Fc receptor. The significance of intimal monocytes for arterial repair and atherogenesis is discussed.

A Bioluminescence Method for the Mapping of Local ATP Concentrations Within the Arterial Wall, With Potential to Assess the In Vivo Situation

According to the anoxemia theory of atherosclerosis, an imbalance between the demand for and supply of oxygen and nutrients in the arterial wall is a key factor in atherogenesis. However, the energy metabolic state of the arterial tissue in vivo is largely unknown. We applied a bioluminescence method, metabolic imaging, to study local ATP concentrations in cryosections of normal pig and atherosclerotic and normal rabbit aorta. Some vessels were subjected to energy metabolic restrictions by incubation at different oxygen and glucose concentrations and others were rapidly frozen in liquid nitrogen to reflect the in vivo situation. Local ATP concentrations and the ATP distribution at a microscale was dependent on oxygen as well as glucose concentrations during incubation. ATP depletion was seen in the mid media of pig aorta in all incubations, but only at low oxygen concentration without glucose in the media of the thinner rabbit aorta. ATP-depleted zones were seen deep in pig media (>750 microm from the lumen) and in rabbit plaques (>300 micrometer+ from the lumen) even at high oxygen (pig 75% O2 and rabbit 21% O2) and glucose concentrations (5.6 mmol/L glucose). This observation probably illustrates an insufficient diffusion of glucose, which highlights the importance of studying the conditions for diffusion not only of oxygen but also of other metabolites in the arterial wall. In rapidly frozen vessels the medial ATP concentration was shown to be 0.6 to 0.8 micromol/g wet weight (both pig and rabbit aorta) and in pig aorta a gradient could be seen indicating higher ATP concentrations at the lumenal side. We propose that metabolic imaging, as applied to snap-frozen tissue, may be used to assess the energy metabolic situation in the arterial wall in vivo. The spatial resolution allows the detection of local variations within the arterial tree. However, steep concentration gradients (eg, near the border of the tissue) will be underestimated. The method may be extended to include determinations of glucose and lactate concentrations and will be used in parallel with an established method to assess hypoxia in the arterial wall in vivo.

Influx and cellular degradation of low density lipoproteins in rabbit aorta determined in an in vitro perfusion system.

The accumulation of 125I-low density lipoprotein (LDL) into normal and atherosclerotic arterial tissue and cellular uptake in arterial cells were studied in an in vitro perfusion system for rabbit aorta. The accumulation of 125I-LDL in normal tissue could be fitted to an inverse exponential function with an initial influx rate of 1.39 nllmg wet weighvhour and an equilibration volume of about 2% of the tissue volume. The influx rate into atherosclerotic plaques was about 10 times faster and the equilibration volume, 50 times higher. In atherosclerotic tissue there was a steep concentration gradient between the plaque and the underlying media. The accumulation of 125I-LDL in the media under plaque and in normal tissue adjacent to plaques was similar to that seen in normal tissue. For studies of cellular uptake of LDL a trace label, 125I-tyraminecellobiose (TC), was used. Normal or atherosclerotic rabbit aorta was perfused in vitro with medium containing 125I-TC-LDL. After perfusion the tissue was digested and the cells were isolated by density gradient centrifugation. Two main cell fractions with characteristics of smooth muscle cells and foam cells, respectively, were obtained. A 70-fold higher uptake was seen in the foam cells. In conclusion, these studies suggest a higher influx rate into atherosclerotic plaques, as well as a high LDL concentration in the plaque, compared with normal tissue or underlying media. We suggest that most of the cellular uptake of LDL in the arterial wall is caused by the foam cells.

A system for long-term perfusion of rabbit aorta in vitro.

In an in vitro system for the perfusion of arterial tissue, the volume of the incubation chamber should be small, and the composition of the medium should be easily modified. The tissue should consist of media and intima only, and the interaction with the medium should occur via the intimal side. Consecutive sampling of the medium and the tissue should be possible. This paper describes a system with these characteristics. Scanning and transmission electron microscopy of the perfused tissue indicated that the endothelium was intact during the first day and that it still covered more than 95% of the surface after 3 days. On the 2nd day, the nonthrombogenic properties of the endothelium were maintained. The medial smooth muscle cells of the inner twothirds of the preparation were viable during the perfusion, while the cells in the outer one-third were dead from the start. Still, the metabolic activity of the tissue was stable, at least during the 2nd day as assessed by the study of DNA, protein, and lipid synthesis, as well as by oxygen consumption. We conclude that the perfusion system presented here might be useful in the study of the interaction between cellular and humoral components of the blood and the arterial wall.

Urokinase Plasminogen Activator Colocalizes with CD25+ Cells in Atherosclerotic Vessels

Proteolytic activity in vascular tissue is necessary for cellular migration, remodelling of extracellular matrix and the development of atherosclerotic lesions. Inflammatory cells, mainly macrophages, are numerous in atherosclerotic plaques and may synthesize and secrete proteolytic enzymes. The principal activator of plasminogen in tissues is urokinase plasminogen activator (u-PA). To determine if an activated phenotype of inflammatory cells colocalizes with local expression of u-PA in atherosclerotic vessels, vascular biopsies from 15 patients with peripheral atherosclerotic disease were analyzed by immunohistochemistry on consecutive sections. Anti-CD68 antibodies were used as markers for macrophages and were positive in 14/15 specimens. Anti-CD25 (interleukin-2 receptor-α) antibodies were used to identify inflammatory cells with an activated phenotype and were positive in 9/14 CD68+ specimens. The same 9 specimens were positive for u-PA. A positive reaction for u-PA was found only in specimens with CD25+ cells. Specimens with positive reactions for all three antibodies were further analyzed with computer-assisted image analysis. The colocalization with u-PA was higher for CD25 compared to CD68 in all specimens. Mean percentage of the u-PA-positive area in regions positive for cellular markers was 52% (SEM 6%) for CD25 and 19% (SEM 5%) for CD68 (p < 0.01). The results indicated that the activation of macrophages in atherosclerotic vessels may modulate local proteolysis and be of importance in plaque development and stability.

Co-Localization of Fibrinolytic Activators and Inhibitors with Macrophages in Atherosclerotic Vessels

The plasmin system is involved in hemostasis and tissue remodelling. The accumulation of plasminogen activators and their inhibitors in atherosclerotic lesions may be due to invasion of inflammatory cells in the vessel wall. High concentrations of macrophages are associated with increased risk of atherosclerotic plaque rupture. By immunohistochemistry on circumferential serial sections of atherosclerotic and healthy vessels the morphological association of plasminogen activators and inhibitors with macrophages was studied. Urokinase plasminogen activator (u-PA), plasminogen activator inhibitor type 2 (PAI-2), and macrophages were mainly expressed within plaques while tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) were also expressed outside plaque lesions. Computer assisted image analysis on diseased vessels showed that regulatory proteins of the fibrinolytic system were found more often in areas positive for macrophages than in other parts of the sections (p < 0.001). u-PA was significantly more defined to areas positive for macrophages than tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) (p < 0.05). Similarly, PAI-2 expression was more associated with macrophage distribution than PAI-1 (p < 0.05). Tumor necrosis factor α (TNFα), an inflammatoric mediator of macrophages, had the same levels of co-localization with macrophages as u-PA and PAI-2. These results suggest that u-PA and PAI-2 might be key factors for inflammatory regulation of plasmin mediated proteolysis in the vessel walls.

Heterogeneous distribution of macrophages, tumour necrosis factor alpha, tissue factor and fibrinolytic regulators in atherosclerotic vessels

OBJECTIVE To assess the representativity of small vascular samples with regard to the expression of six important factors in atherosclerotic vessels. MATERIALS Circumferential frozen sections of iliac and femoral arteries (n = 9) from patients undergoing reconstructive bypass surgery. METHODS Immunohistochemistry with antibodies against macrophages (CD68), tumour necrosis factor alpha, tissue factor, tissue plasminogen activator, urokinase plasminogen activator and plasminogen activator inhibitor type 1. The distribution of these antigens was characterised by computer assisted image analysis. Antigen positive area in randomly chosen samples of varying size was compared with antigen positive area in the whole vessel transections. RESULTS Marked heterogeneity was found with respect to the expression of these factors in atherosclerotic vessels. The representativity of samples was highly dependent on the size of the samples. PAI-1 was more evenly expressed compared to the other antigens. CONCLUSIONS Estimates of the expression of these factors based on small samples from atherosclerotic arteries are unreliable.

Melagatran attenuates fibrin and platelet deposition in a porcine coronary artery over-stretch injury model.

&NA; Melagatran attenuates fibrin and platelet deposition in a porcine coronary artery over‐stretch injury model Melagatran is the active form of the oral direct thrombin inhibitor, ximelagatran. The purpose of this study was to compare the effects of different doses of melagatran with heparin or placebo on platelet deposition and relative fibrin content after coronary angioplasty in pigs. After 125I‐labelled fibrinogen and autologous 111Indium‐labelled platelets had been infused a balloon injury was performed in the left anterior descending and the right coronary arteries. Pigs were randomized to receive either heparin 200 IU/kg bolus plus 20 IU/kg per h infusion (n = 7); melagatran 1 mg/kg bolus plus 0.33 mg/kg per h infusion (n = 7); melagatran bolus 0.5 mg/kg plus 0.17 mg/kg per h infusion (n = 7); melagatran 0.15 mg/kg bolus plus 0.05 mg/kg per h infusion (n = 6) or saline (n = 4). Seventy‐five minutes after the angioplasty, the pigs were euthanized and the injured vessel segments were measured in a gamma counter. Compared with placebo, platelet deposition and relative fibrin content were reduced after both heparin and melagatran, in the latter case with a dose‐response relationship. Melagatran reduced platelet deposition and relative thrombus size in a dose‐dependent manner when compared with placebo after coronary angioplasty in pigs. No statistically significant difference between melagatran and heparin was found. Blood Coagul Fibrinolysis 14:235‐241