Guy M. Chisolm

Endothelial and smooth muscle cells alter low density lipoprotein in vitro by free radical oxidation.

Our purpose was to determine whether the action of oxidative free radicals released by endothelial cells and vascular smooth muscle cells grown in culture could be responsible for certain modifications to low density lipoprotein (LDL). In these experiments we showed that after a 48-hour incubation with human umbilical vein endothelial cells or bovine aortic smooth muscle cells, human LDL: 1) became oxidized, as evidenced by reactivity to thiobarbituric acid; 2) lost variable amounts of sterol relative to protein (up to 20%); 3) had an increased relative electrophoretic mobility (by 30% to 70%); and 4) became toxic to proliferating fibroblasts. None of these changes occurred after a 48-hour incubation with confluent fibroblasts or bovine aortic endothelial cells, and all could be virtually prevented by the presence of butylated hydroxytoluene or other free radical scavengers. The results suggest that cells modifying LDL do so in part by an oxidation of LDL subsequent to cellular generation of free radicals.

Lipoprotein oxidation and lipoprotein-induced cytotoxicity.

The results of this study Indicate that when human VLDL or LDL Is prepared under conditions allowing oxidation, such oxidation renders the molecular complexes highly toxic to human skin flbroblasta growing In culture. The cytotoxicity can be predicted by assaying for the presence of thlobarbiturlc acld-reactlng substances on the lipoprotein. However, malondialdehyde, which reacts with thiobarblturlc acid and Is known to be Injurious to cells, was not cytotoxic In the same experimental system when dissolved In culture medium or covalently bound to non-toxic LDL. The toxic agent(s) on oxidized LDL Is(are) located In a llpld-extractable moiety. Since lipld peroxides and oxidized sterols can occur in vivo under various pathological conditions, the cytotoxicity of these llpoprotein-assoclated substances observed In vitro may be related to certain manifestations of these conditions.

Monocytes and Neutrophils Oxidize Low Density Lipoprotein Making It Cytotoxic

Free radicals are believed to be involved in leukocyte induced tissue injury. The present studies were performed to determine whether low density lipoprotein (LDL) might serve as a mediator of tissue injury after leukocyte induced free radical oxidation of LDL. Our results show that incubation of LDL with monocytes or polymorphonuclear leukocytes (PMN) leads to oxidation of the lipoprotein rendering it toxic to proliferating fibroblasts. Monocyte activation enhances these effects. Butylated hydroxytoluene (BHT), vitamin E (vit E) and glutathione (GSH) virtually prevent the oxidation of LDL and the formation of cytotoxic LDL, indicating that these alterations are mediated by leukocyte‐derived free radicals. This is the first demonstration that short‐lived free radicals emanating from phagocytic cells could mediate cell injury through the action of a stable cytotoxin formed by the oxidation of LDL. The fact that lipoproteins can transfer a cytotoxic effect from leukocytes to proliferating cells reveals a pathway for cell destruction which may have implications in atherosclerotic plaque progression, macrophage mediated toxicity to tumor cells and tissue injury by inflammatory processes.

LDL-induced cytotoxicity and its inhibition by HDL in human vascular smooth muscle and endothelial cells in culture

Human aortic medial smooth muscle cells (SMC) and umbilical vein endothelial cells (EC) in culture were exposed to various concentrations of plasma low density (LDL) and high density (HDL) lipoproteins prepared from normolipemic donors in order to assess their effects on cell growth. So that the effects of each lipoprotein could be evaluated separately and in combination, lipoproteins were added to culture medium containing lipoprotein deficient serum (LPDS, d greater than 1.25 g/ml at a protein concentration of 4.5 mg/ml of medium). The addition of LDL at cholesterol concentrations of 160 microgram/ml of culture medium, resulted in significant reductions in both the number of SMC and EC cells per dish within 3 days of exposure (P less than 0.001, SMC; P less than 0.01, EC), when compared with LPDS controls and the starting cell numbers. This cytotoxic phenomenon was dose-related, and only at LDL cholesterol concentrations equal to or below 50 microgram/ml were no marked changes observed. In contrast, HDL at all concentrations tested produced no such deleterious effects. Autoradiographic assessment of DNA synthesis confirmed these findings. After 48 h of continuous exposure to tritiated thymidine, labeling indexes reached much lower plateaus in the LDL-treated groups.

Modification of human serum low density lipoprotein by oxidation--characterization and pathophysiological implications.

Plasma low density lipoprotein (LDL) can undergo free radical oxidation either catalyzed by divalent cations, such as Cu2+ or Fe2+ or promoted by incubation with cultured cells such as endothelial cells, smooth muscle cells and monocytes. The content of vitamin E, beta-carotene and unsaturated fatty acids is decreased in oxidized LDL. A breakdown of apolipoprotein-B (apoB), hydrolysis of the phospholipids, an increase of thiobarbituric acid reactive substances and the generation of aldehydes also occur. Changes in the ratio of lipid to protein, the electrophoretic mobility and the fluorescent properties have also been reported to accompany oxidation of this lipoprotein. The functional changes of oxidized LDL include its recognition by the scavenger receptor on macrophages, its cytotoxicity especially to proliferating cells, its chemotactic properties with respect to monocyte-macrophages and its regulation of platelet-derived growth factor-like protein (PDGFc) production by endothelial cells. In this article we summarize some of the contributions to this topic and present speculations relating oxidized LDL to pathological conditions such as atherosclerosis.

Restenosis After Experimental Angioplasty: Intimal, Medial, and Adventitial Changes Associated With Constrictive Remodeling

Predicting and preventing arterial restenosis after angioplasty has failed despite considerable research into mechanisms and techniques. We examined the roles of chronic constriction, neointimal-medial growth, and adventitial changes in restenosis in atherosclerotic rabbits. Angioplasty was performed on femoral artery lesions 4 weeks after lesion induction by air drying and cholesterol-supplemented diet. Angiographic and histological evaluation was conducted 3 to 4 weeks after angioplasty. The angiographic minimum luminal diameter (MLD) increased from 1.31 +/- 0.21 to 1.73 +/- 0.41 mm after angioplasty. Loss in MLD by 3 to 4 weeks was 0.95 +/- 0.64 mm. Initial gain and late loss correlated (P = .008). Late residual stenosis, defined histologically as the difference between the luminal areas of a proximal reference site and lesion site normalized by the luminal area of the reference site, was 52 +/- 32%. Histological indices of chronic constriction, neointimal-medial growth, and adventitial growth were defined on the basis of the areas of these arterial wall layers at the lesion site relative to the reference site. Another parameter defined as the ratio of adventitial area to the area of intima+media at the lesion site allowed evaluation of the relative importance of these layers. Surprisingly, late residual stenosis correlated with chronic constriction (P = .0003) but not with neointimal-medial growth or adventitial growth. The ratio of adventitial area to the area of intima+media at the lesion site also correlated with chronic constriction (P = .01). These findings suggest that factors related to arterial remodeling rather than neointimal-medial growth may dominate the response to angioplasty.

Intact human ceruloplasmin oxidatively modifies low density lipoprotein.

Ceruloplasmin is a plasma protein that carries most of the copper found in the blood. Although its elevation after inflammation and trauma has led to its classification as an acute phase protein, its physiological role is uncertain. A frequently reported activity of ceruloplasmin is its ability to suppress oxidation of lipids. In light of the intense recent interest in the oxidation of plasma LDL, we investigated the effects of ceruloplasmin on the oxidation of this lipoprotein. In contrast to our expectations, highly purified, undegraded human ceruloplasmin enhanced rather than suppressed copper ion-mediated oxidation of LDL. Ceruloplasmin increased the oxidative modification of LDL as measured by thiobarbituric acid-reacting substances by at least 25-fold in 20 h, and increased electrophoretic mobility, conjugated dienes, and total lipid peroxides. In contrast, ceruloplasmin that was degraded to a complex containing 115- and 19-kD fragments inhibited cupric ion oxidation of LDL, as did commercial preparations, which were also degraded. However, the antioxidant capability of degraded ceruloplasmin in this system was similar to that of other proteins, including albumin. The copper in ceruloplasmin responsible for oxidant activity was not removed by ultrafiltration, indicating a tight association. Treatment of ceruloplasmin with Chelex-100 removed one of seven copper atoms per molecule and completely blocked oxidant activity. Restoration of the copper to ceruloplasmin also restored oxidant activity. These data indicate that ceruloplasmin, depending on the integrity of its structure and its bound copper, can exert a potent oxidant rather than antioxidant action on LDL. Our results invite speculation that ceruloplasmin may be in part responsible for oxidation of LDL in blood or in the arterial wall and may thus have a physiological role that is quite distinct from what is commonly believed.

Rat phospholipid-hydroperoxide glutathione peroxidase. cDNA cloning and identification of multiple transcription and translation start sites.

Phospholipid-hydroperoxide glutathione peroxidase (PhGPx) is a selenoenzyme that reduces hydroperoxides of phospholipid, cholesterol, and cholesteryl ester. Previous studies suggested that both the mitochondrial and nonmitochondrial forms of PhGPx are 170 amino acids long. In this study, we isolated a full-length cDNA clone encoding rat testis PhGPx. Based on sequence analysis, the cDNA encodes a protein of 197 amino acids, with translation initiating at AUG. The additional 27 amino acids at the N terminus contain the features of a mitochondrial targeting sequence. In vitro translation of the full-length PhGPx mRNA initiated predominantly at AUG. However, translation initiated at AUG when AUG was deleted. An RNase protection assay was used to map the 5′-ends of PhGPx mRNAs in rat tissues. We identified two major windows of transcription initiation that are tissue-specific. Rat testis predominantly expresses larger transcripts that encode the 197-amino acid protein containing the potential mitochondrial targeting signal. The predominant smaller transcripts in somatic tissues lack AUG and encode a 170-amino acid protein, which may represent the nonmitochondrial forms of PhGPx. Our results suggest that the use of alternative transcription and translation start sites determines the subcellular localization of PhGPx in different tissues.

Oxidized LDL-Induced Injury and Apoptosis in Atherosclerosis Potential Roles for Oxysterols

The cell injury caused by oxidized lipoproteins was among the first findings that led to the theory that it is the oxidation of low-density lipoprotein (LDL), not just LDL concentration, that leads to arterial disease. Voluminous studies have now revealed that oxidized lipoproteins and their constituents can induce numerous effects on cells that can be construed to be atherogenic. Cell injury is but one of these, and it is these injurious effects that are the focus of this brief review. Cell injury and death appear to play multiple roles in lesion development and the toxic lipid constituents of oxidized lipoproteins, including a variety of oxysterols, are candidates for the in vivo effectors of this cytotoxicity. Recent studies have focused on the mechanisms of oxidized lipoprotein-induced cell death, whether the cells die by apoptosis or necrosis, and the identities of the toxins that induce injury. Understanding the roles of these agents in lesion development could lead to therapies that modulate cell death and inhibit lesion formation.

The oxidation of lipoproteins by monocytes-macrophages. Biochemical and biological mechanisms

The oxidation of lipoproteins has been proposed as a biological process that initiates and accelerates arterial lesion development (1–5). Oxidized lipoproteins accumulate in lesions (6) and may form at other inflammatory sites. Whether the oxidized lipoprotein is an initiator or accelerator of disease is the subject of speculation, debate, and intensive study. Various cellular and biochemical mediators of lipoprotein oxidation in vivo have been proposed, but none has yet been proven to be responsible. Two decades ago we demonstrated that low density lipoprotein (LDL), the plasma level of which correlates with the risk of atherosclerosis, could injure endothelial cells (ECs) in culture (7). The capacity of LDL to injure cells was directly related to the level of LDL oxidation, and we speculated on a possible role for oxidized LDL-mediated endothelial injury in atherogenesis (8, 9). Contemporaneously, Dr. Daniel Steinberg’s group (10, 11) demonstrated that LDL exposed to cultured ECs was altered such that it became a ligand for scavenger receptors. In 1984, both Steinberg’s group and ours (12, 13) demonstrated that the “EC-modified LDL” they had characterized and the “oxidized LDL” we had described were the same entity. Their report highlighted the macrophage recognition of the EC-oxidized lipoprotein, and ours the capacity of EC or smooth muscle cell (SMC)-oxidized LDL to injure cells. These papers introduced the concept that reactive oxygen species from vascular cells could transform LDL, causing it to exhibit dramatically altered composition and atherogenic properties. The first demonstration that certain leukocyte populations could oxidize LDL employed human neutrophils and activated populations of adherent human monocytes, cells well known to generate abundant reactive oxygen species in vitro and in vivo (14). The identity of the cells responsible for the oxidation of LDL that accumulates in lesions is uncertain. Although it is well known that free ferrous or cupric ions catalyze lipid peroxidation reactions in vitro, the presence of free metal ions in vivo is doubted (15). Multiple mechanisms exist in vivo for binding free transition metal ions, rendering them redox-inactive (15–17). In this minireview, we take the position that monocyte-derived macrophages are likely candidates to mediate the in vivo oxidation of lipoproteins, because they are prominent in arterial lesions, known to generate activation-dependent reactive oxygen species, and, unlike EC and SMC (12, 18), capable in vitro of LDL oxidation in media free of metal ion additives. In vitro LDL appears to be oxidized extracellularly without interaction with the LDL receptor (19–21). There are multiple potential pathways through which monocytes-macrophages may promote extracellular LDL oxidation. In this review we evaluate cellular mechanisms (both enzymatic and non-enzymatic) for LDL oxidation. We use the term “monocyte-macrophage” as a shorthand reference to in vitro studies performed on isolated monocytes, macrophages, and monocyte-like cell lines.