Atherosclerotic Arterial Intima Research Articles

Editorial introductions

Current Opinion in Lipidology was launched in 1990. It is part of a successful series of review journals whose unique format is designed to provide a systematic and critical assessment of the literature as presented in the many primary journals. The field of lipidology is divided into six sections that are reviewed once a year. Each section is assigned a Section Editor, a leading authority in the area, who identifies the most important topics at that time. Here we are pleased to introduce the Section Editor for this issue. Section Editors Petri T. KovanenPetri T. KovanenDr Kovanen received his medical degree in 1970 from the University of Basle, Switzerland, and his M.D. Ph.D degree in 1975 from the University of Helsinki, Finland. He conducted postdoctoral work (1976 – 1980) with Nobel Laureates Joseph L. Goldstein and Michael S. Brown in the Departments of Molecular Genetics and Internal Medicine at the University of Texas, Health Science Center at Dallas, USA. After his return to Finland, he completed his residency in Internal Medicine at the University of Helsinki (head, the late Professor Esko Nikkilä). In 1984 he was appointed Scientific Director of Wihuri Research Institute, Helsinki, and currently serves as Director of the Institute. His main research interests focus on the pathogenesis of atherosclerosis, both on early atherogenesis with emphasis on the mechanisms of extracellular lipid accumulation, and on late atherogenesis with emphasis on the generation of vulnerable plaques. He has been exploring the role of mast cells, rediscovered invaders of the atherosclerotic arterial intima, in atherogenesis and its clinical complications. Dr Kovanen has published numerous scientific articles, reviews, and book chapters on topics dealing with the pathophysiology of atherosclerosis, particularly on proteolytic and lipolytic modifications of LDL and HDL. The leading theme of his work has been exploration of the roles of mast cells as potent effector cells in atherogenesis. Dr Kovanen has served as Editor-in-Chief of Annals of Medicine, and as Editorial Board Member of Arteriosclerosis, Thrombosis, and Vascular Biology. He has also served as a Council Member of the Scandinavian Society for Atherosclerosis Research and Councillor of the European Vascular Biology Organization. He is past President of the Finnish Atherosclerosis Society, and is currently Secretary of the European Atherosclerosis Society, and also chairs its Scientific Committee. Andrew NewbyAndrew NewbyAndrew Newby is British Heart Foundation Professor of Vascular Cell Biology. He graduated in Natural Sciences (Biochemistry) from Cambridge and studied for a PhD with Professor CN Hales FRS. He then worked on adenylate cyclase with Nobel Laureate Martin Rodbell at the NIH in Bethesda, Maryland. Afterwards, he held a Beit Memorial Fellowship in Cambridge where he elucidated the metabolic pathways responsible for production of the cardioprotective metabolite, adenosine. Subsequently, he was successively Non-clinical Lecturer, Senior Lecturer, Reader and Professor in Cardiff. While continuing to work on adenosine, he contributed to the identification of the endothelium-derived relaxing factor as nitric oxide. He is most know however, for discovering a role for matrix degrading metalloproteinases in vascular smooth muscle cell migration and proliferation in vein grafts, after angioplasty and in atherosclerosis. His elucidation of the inflammatory basis of metalloproteinase production is continuing to shed light on the role of inflammation in plaque rupture and myocardial infarction. He was one of the first vascular biologists to use of adenovirus-mediated gene transfer which he now combines with other post-genome technologies. This work has led to more than 150 peer-reviewed research papers and 30 reviews, which have collectively attracted 8000 citations (H>57). It was continuously supported by UK programme grants for the past 20 years. Prof. Newby was also Co-director of the EC-funded European Vascular Genomics Network. Prof. Newby has served on grants committees of the major UK Research Councils and Charities and also reviewed grants for Belgian, Dutch, French, German and other overseas bodies. His Editorial Boards include Atherosclerosis, ATVB and Cardiovascular Research. Prof. Newby has been an EAS member for many years and recently served on the Programme Committee for the Helsinki and Gothenburg congresses. He has more than 20 years involvement with the European Society of Cardiology, being a founder member and later Chair of the working group Pathogenesis of Atherosclerosis, a member of the Congress Programme Committee and Chairman of the Council on Basic Cardiovascular Science. He now dedicates himself to running the biennial ESC Summer Schools in Cardiovascular Biology. Perhaps most significantly he re-launched and was President of the European Vascular Biology Organisation from 2006 to 2010. Website: http://www.bristol.ac.uk/clinicalsciencesouth/people/personpages/a.newby.html.

Enhanced extracellular lipid accumulation in acidic environments

Binding of apolipoprotein B-100-containing lipoproteins (VLDL, IDL, and LDL) to proteoglycans and modifications of the lipoproteins, whether bound or unbound, are key processes in atherogenesis. The complex interplay between binding and modification has been studied at neutral pH conditions. It has been demonstrated that during atherogenesis the extracellular pH of the lesions decreases. We summarize findings suggesting that lipoprotein binding and modification are enhanced at acidic pH. Many enzymes found in the arterial intima, such as secretory sphingomyelinase and cathepsins, are able to hydrolyze lipoproteins in vitro. These enzymes function optimally at slightly acidic pH (pH 5.5-6.5), and are likely to act on lipoproteins optimally in the acidic plaque areas. Also, the ability of human aortic proteoglycans to bind native VLDL, IDL, and LDL is dramatically increased at acidic pH; this binding can be further increased if these apolipoprotein B-100-containing particles are hydrolytically modified. Recent in-vitro findings suggest that in areas of atherosclerotic arterial intima where the extracellular pH is decreased, binding of apolipoprotein B-100-containing lipoproteins to proteoglycans and modification of the lipoproteins by acidic enzymes are enhanced. The pH-induced amplification of these processes will lead to enhanced extracellular accumulation of lipoproteins and accelerated progression of the disease.

Decrease in pH Strongly Enhances Binding of Native, Proteolyzed, Lipolyzed, and Oxidized Low Density Lipoprotein Particles to Human Aortic Proteoglycans

Binding of low density lipoprotein (LDL) to proteoglycans and modification of LDL are key processes in atherogenesis. Recently, it has been demonstrated that during atherogenesis the extracellular pH of atherosclerotic lesions decreases. We have examined the effect of the decreased pH on the binding of LDL to human aortic proteoglycans. The binding of native, oxidized, proteolyzed (alpha-chymotrypsin-treated), or lipolyzed (sphingomyelinase- or phospholipase A(2)-treated) LDL particles to proteoglycans were measured in microtiter well assays at pH 5.5-7.5. We found that the lower the pH, the higher the amount of binding of LDL to proteoglycans. At the lowest pH tested (pH 5.5), the amounts of proteoglycan-bound native, proteolyzed, sphingomyelinase-, and phospholipase A(2)-treated LDL were 20-, 23-, 30-, and 37-fold higher, respectively, than at pH 7.5. Interestingly, although oxidized LDL failed to bind to proteoglycans at neutral pH, there was significant binding at acidic pH. Binding of native and modified LDL to proteoglycans at pH 5.5 was blocked by 1 m NaCl, indicating that at neutral pH LDL binds to proteoglycans via ionic interactions. Inhibition of this binding by acetylation and cyclohexanedione treatment of LDL showed that the positively charged amino acids of apolipoprotein B-100, lysine, and arginine, respectively, mediated the ionic interaction. Taken together, our results suggest that in areas of atherosclerotic arterial intima where the extracellular pH decreases, retention of LDL by proteoglycans is enhanced, leading to extracellular accumulation of LDL and progression of the disease.

Expression of keratinocyte growth factor receptor (KGFR/FGFR2 IIIb) in vascular smooth muscle cells

Keratinocyte growth factor receptor (KGFR), also known as fibroblast growth factor receptor (FGFR)2 IIIb, is located in many types of epithelial cells and is activated by four known ligands (FGF-1, FGF-3, FGF-7 (also known as KGF) and FGF-10) that are predominantly synthesized by mesenchymal cells. In the early stage of atherosclerosis, vascular smooth muscle cells (VSMC) transform from a contractile to a synthetic phenotype, proliferate and migrate into the intima. Previously, FGF-7 mRNA expression was reported in VSMC, but KGFR mRNA was not detected. In the present study, we attempted to determine whether KGFR is localized in VSMC cultured from rat aorta and VSMC in human normal and atherosclerotic coronary arteries. Expression of KGFR mRNA and its protein was detected in cultured rat VSMC by reverse transcription-polymerase chain reaction and western blot analysis, respectively. Immunohistochemically, KGFR was localized in the VSMC of the outer layer of the media in normal human coronary arteries. Furthermore, it was localized in the VSMC of the media and thickened intima of atherosclerotic arteries. Recombinant FGF-7 and/or FGF-10 proteins stimulated the growth of cultured rat VSMC. These findings indicate that KGFR localized in VSMC may contribute to the proliferation of VSMC in normal and atherosclerotic arteries.

Plasmin and Kallikrein Reduce HDL-Induced Cholesterol Efflux from Foam Cells

Arterial intima contains metabolically active factors such as proteases, which may act on high-density lipoprotein (HDL) and impair its ability to accept cholesterol. In this study we treated human HDL3 with human plasmin and human plasma kallikrein, two proteases also found in the human arterial intima, in order to study their effect on the ability of HDL3 to promote cholesterol efflux from human macrophage foam cells. After exposure to plasmin or plasma kallikrein for 15 min, HDL3 showed a decrease of about 60% in its ability to promote cholesterol efflux from the macrophage foam cells. SDS–PAGE analysis of the degraded HDL3 particles showed that plasmin had generated cleavage products less than 15 kDa in size and plasma kallikrein had generated a major product of about 19 kDa. However, there was only a slight loss of intact apolipoproteins, suggesting degradation of a small subpopulation of HDL3 particles. Agarose gel electrophoresis showed that a decrease in cholesterol efflux was accompanied by total loss of the HDL3 with preβ-mobility, but no apparent change in those with α mobility. These results suggest that the presence of active plasmin or plasma kallikrein in the atherosclerotic arterial intima promotes atherogenesis by blocking cholesterol efflux from macrophage foam cells.

Immunohistochemical analysis of the plasminogen activation system and the matrix metalloproteinases in normal and atherosclerotic human vessels

In this study we examined the localization of the plasminogen activation system and the matrix metalloproteinases in normal human arteries, arteries with diffuse intimal thickenings and atherosclerotic arteries. We found that MMP-1, MMP-2, MMP-3, TIMP-1 and TIMP-2 did not stain differently in normal arteries, arteries with intimal thickening and atherosclerotic arteries. MMP-9 staining was, however, increased in atherosclerotic arteries as compared to normal arteries and arteries with diffuse intimal thickening. For u-PA there were no differences between normal arteries, arteries with intimal thickening and atherosclerotic arteries. t-PA staining was decreased in the media of atherosclerotic arteries as compared to both normal and intima-thickened arteries. In contrast, PAI-1 staining was elevated in the intima of atherosclerotic arteries as compared to the media. While u-PAR was elevated in the intima of arteries with intimal thickening, staining was weaker in the intima of atherosclerotic arteries, as compared to the intima of normal arteries. These results suggest that both the plasminogen activation system and the MMP system might be involved in the process of atherosclerosis most likely in different steps and stages in this process.

Fusion of proteolyzed low-density lipoprotein in the fluid phase: a novel mechanism generating atherogenic lipoprotein particles.

During atherogenesis, lipid droplets appear in the extracellular space of the arterial intima. We previously observed generation of lipid droplets on the surface of exocytosed mast cell granules when granule neutral proteases degraded the granule-bound LDL particles and the particles became unstable and fused [Kovanen, P.T., & Kokkonen, J.O. (1991) J. Biol. Chem. 266, 4430-4436]. We have now extended our studies to the fluid phase and examined the effects of several proteases (trypsin, alpha-chymotrypsin, Pronase, plasmin, kallikrein, and thrombin) all known for their ability to cleave the apolipoprotein B-100 component (apoB-100) of LDL. The fused LDL particles were separated from unfused particles by gel filtration or by density gradient ultracentrifugation. Proteolytic degradation of LDL with trypsin, alpha-chymotrypsin, or Pronase led to fragmentation of apoB-100 and release of the fragments from the LDL particles and triggered particle fusion. In contrast, proteolytic degradation of LDL with plasmin, kallikrein, or thrombin, which also led to fragmentation of apoB-100 but not to release of fragments, did not trigger particle fusion. With advancing degradation of apoB-100, particles having progressively lower densities and larger sizes were generated. Thus, after incubation for 24 h with alpha-chymotrypsin (apoB-100:alpha-chymotrypsin mass ratio 10:1) 40% of the apoB-100 was degraded and about 30% of the LDL particles had fused and reached diameters of up to 70 nm and densities ranging from 1.020 to < 1.005 g/mL. When the proteolyzed LDL particles, both unfused and fused, were incubated with macrophages, only those particles that had undergone fusion were ingested and converted into intracellular cholesteryl ester droplets. Thus proteolysis of LDL with release of apoB-100 fragments renders the particles sufficiently unstable to fuse and thus to become liable to ingestion by macrophages. Since the fused LDL particles resemble the extracellular lipid droplets in the atherosclerotic arterial intima and generate foam cells in vitro, these findings support the idea that proteolytic fusion of LDL is an atherogenic process.

Proteolysis and Fusion of Low Density Lipoprotein Particles Strengthen Their Binding to Human Aortic Proteoglycans

Lipid droplets resembling those seen in the extracellular space of the arterial intima were generated in vitro when granule proteases of rat serosal mast cells degraded the apolipoprotein B-100 (apoB-100) component of granule-bound low density lipoprotein (LDL), and the particles fused on the granule surface (Paanenen, K., and Kovanen, P. T. (1994) J. Biol. Chem. 269, 2023-2031). Moreover, the binding of the fused particles to the heparin proteoglycan component of the granules was found to be strengthened. We have now treated LDL particles with alpha-chymotrypsin and examined the strength with which the proteolytically modified LDL binds to human aortic proteoglycans on an affinity column. We found that chymotryptic degradation of the LDL particles triggered particle fusion. The higher the degree of proteolytic degradation, the higher were the degree of fusion and the strength of binding to the aortic proteoglycans. Separation of the proteolyzed particles by size exclusion chromatography into two fractions, unfused and fused particles, and analysis of their binding strengths revealed that not only the fused but also the unfused proteolyzed particles bound more tightly to the proteoglycans than did the native LDL particles. To investigate the mechanism underlying this increase in binding strength, we attached [13C]dimethyl groups to the lysines and used NMR spectroscopy to quantify the active lysine residues of apoB-100, which are thought to be located in basic areas of apoB-100 and involved in binding of LDL to proteoglycans. Analysis of the 13C-labeled particles showed that, despite loss of apoB-100 fragments from the particles, the number of active lysine residues in the unfused proteolyzed particles had not decreased. In the fused proteolyzed particles, the number of active lysine residues was markedly increased. Thus, proteolytic fusion appears to increase the number of basic domains of apoB-100, which would explain the observed increase in the strength of binding of the modified LDL particles to arterial proteoglycans. Since the fused particles resemble the small lipid droplets found in the atherosclerotic arterial intima, this LDL modification offers a plausible mechanism for the focal accumulation of lipid droplets in the extracellular proteoglycan matrix during atherogenesis.

Evidence of Increased Fibrinolytic Activity in the Intima of Atherosclerotic Arteries

Evidence of Increased Fibrinolytic Activity in the Intima of Atherosclerotic Arteries

Increased type 1 plasminogen activator inhibitor gene expression in atherosclerotic human arteries.

Decreased fibrinolytic capacity has been suggested to accelerate the process of arterial atherogenesis by facilitating thrombosis and fibrin deposition within developing atherosclerotic lesions. Type 1 plasminogen activator inhibitor (PAI-1) is the primary inhibitor of tissue-type plasminogen activator and has been found to be increased in a number of clinical conditions generally defined as prothrombotic. To investigate the potential role of this inhibitor in atherosclerosis, we examined the expression of PAI-1 mRNA in segments of 11 severely diseased and 5 relatively normal human arteries obtained from 16 different patients undergoing reconstructive surgery for aortic occlusive or aneurysmal disease. Densitometric scanning of RNA (Northern) blot autoradiograms revealed significantly increased levels of PAI-1 mRNA in severely atherosclerotic vessels (mean densitometric value, 1.7 +/- 0.28 SEM) compared with normal or mildly affected arteries (mean densitometric value, 0.63 +/- 0.09 SEM; P less than 0.05). In most instances, the level of PAI-1 mRNA was correlated with the degree of atherosclerosis. Analysis of adjacent tissue sections from the same patients by in situ hybridization demonstrated an abundance of PAI-1 mRNA-positive cells within the thickened intima of atherosclerotic arteries, mainly around the base of the plaque. PAI-1 mRNA could also be detected in cells scattered within the necrotic material and in endothelial cells of adventitial vessels. In contrast to these results, PAI-1 mRNA was visualized primarily within luminal endothelial cells of normal-appearing aortic tissue. Our data provide initial evidence for the increased expression of PAI-1 mRNA in severely atherosclerotic human arteries and suggest a role for PAI-1 in the progression of human atherosclerotic disease.

Photofrin IIが動脈硬化部位におけるレーザー蒸散能に与える影響

Fluorescence misroscopy and light microscopy were performed to investigate the affinity of photofrin II for atherosclerotic lesions and the effect of photofrin II in atherosclerotic rabbits aortas when vessels were dissected and submitted to laser radiation. Atherosclerotic lesions in rabbits were induced by 2%cholesterol diet and damage by Fogarty balloon catheter. Atherosclerotic rabbits were divided into two groups. Group P comprised atherosclerotic rabbits given photofrin II (5mg/kg) intravenously 24 hours before sacrifice. Group C comprised atherosclerotic rabbits that were not given photofrin II. Laser radiation was delivered with Argon Laser for 20 seconds (output 514.5nm) and the power was 500mW, 700mW, and 900mW. The photoradiated sites were sectioned and stained with Hematoxylin Eosin for light microscopy. Atherosclerotic lesions were frozen quickly and sectioned for fluorescence microscopy. Photofrin II was selectively localized within the intima of atherosclerotic arteries, and in Group P atherosclerotic lesions were vaporized by lower energy level than in Group C. This localization may sensitize atheroma to Argon Laser.

Lipid Peroxidation and atherosclerosis

Atherosclerosis and its clinical sequelae are among the most important of human cardiovascular diseases. Abnormal amounts of lipid are usually present in the thickened intima of atherosclerotic arteries. The notion that lipid peroxidation and polymerization occur in these lipid deposits is based largely on the fact that ceroid pigments have been demonstrated in atherosclerotic plaques. The detection of cholesteryl hydroxyoctadecadienoic acids in lipids of atheromas is suggestive evidence of lipoperoxidation; detection of lipoperoxides as such is technically difficult and the results to date are inconclusive. Studies of arteries from man and animals indicate that ceroid pigments occur late in the disease process and are not likely to be initiators of atherosclerosis. It is possible that hydroperoxides or intermediate free radiclals participate in the early stages of atherogenesis, but this theory is without conclusive support. Ceroid has been implicated as an inflammatory or sclerosing agent in arteries, p...

Incorporation of 14-C-labeled acetate into lipid by isolated foam cells and by atherosclerotic arterial intima.

The synthesis of lipid by rabbit atherosclerotic intima incubated in vitro has been investigated and compared with that of foam cells isolated from intimal lesions. In the atherosclerotic arterial intima, 14 C-labeled acetate is readily incorporated into the lipid fraction, most of the label being found in the phos-pholipid and cholesterol ester fatty acid fractions. Minimal incorporation into triglyceride, fatty acid, and cholesterol occurred. The major phospholipid synthesized was lecithin but significant incorporation into phosphatidyl inositol, phosphatidyl ethanolamine, and sphingomyelin also took place. The fatty acids synthesized were predominantly saturated in the phospholipid fraction. More monounsaturated fatty acids were present in the cholesterol ester fatty acids, but in both moieties little polyunsaturated fatty acids were labeled by the acetate. Isolated foam cells were also shown to incorporate 14 C-labeled acetate into both phospholipid and cholesterol ester fatty acids. The foam cells, however, incorporated a significantly higher proportion of acetate into cholesterol ester than did the whole intima. The fatty acids synthesized by the foam cells were more polyunsaturated in both the phospholipid and the cholesterol ester fraction than was the case with the whole intima. The relevance of these findings to the origin of lipid in the atherosclerotic lesion is discussed.