Günther Jürgens

The role of lipid peroxidation and antioxidants in oxidative modification of LDL.

The purpose of this study is to provide a comprehensive survey on the compositional properties of LDL (e.g., lipid classes, fatty acids, antioxidants) relevant for its susceptibility to oxidation, on the mechanism and kinetics of LDL oxidation, and on the chemical and physico-chemical properties of LDL oxidized by exposure to copper ions. Studies on the occurrence of oxidized LDL in plasma, arteries, and plaques of humans and experimental animals are discussed with particular focus on the use of poly- and monoclonal antibodies for immunochemical demonstration of apolipoprotein B modifications characteristic for lipid peroxidation. Apart from uptake of oxidized LDL by macrophages, studies describing biological effects of heavily or minimally oxidized LDL are only briefly addressed, since several reviews dealing with this subject were recently published. This article is concluded with a section on the role of natural and synthetic antioxidants in protecting LDL against oxidation, as well as some previously unpublished material from our laboratories.

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.

Modification of human low-density lipoprotein by the lipid peroxidation product 4-hydroxynonenal.

The effects of the lipid peroxidation product 4-hydroxynonenal on freshly prepared human low-density lipoprotein (LDL) were studied. At a fixed LDL concentration (5.7 mg/ml) the amount of 4-hydroxynonenal incorporated into the LDL increased with increasing aldehyde concentration from 28-30 (0.2 mM) to 140 (1 mM) mol per mol LDL, whereas at a fixed aldehyde concentration (0.2 mM) its incorporation into LDL decreased with increasing LDL concentration from 48 (1 mg LDL/ml) to 26 (12 mg LDL/ml) mol 4-hydroxynonenal bound per mol LDL. Of the total hydroxynonenal taken up 78% was bound to the protein and 21% to the lipid moiety; the remaining 1% was dissolved as free aldehyde in the lipid fraction. Amino acid analysis of the apolipoprotein B revealed that 4-hydroxynonenal attacks mainly the lysine and tyrosine residues and to a lesser extent also serine, histidine and cysteine. Treatment of LDL with 4-hydroxynonenal results in a concentration-dependent increase of the negative charge of the LDL particle as evidenced by its increased electrophoretic mobility. Moreover, 4-hydroxynonenal treatment leads to a partial conversion of the apolipoprotein B-100 into higher molecular weight forms most probably apolipoproteins B-126 and B-151. Compared to malonaldehyde, 4-hydroxynonenal exhibits a much higher capacity to modify LDL and it is therefore believed that this aldehyde is a more likely candidate for being responsible for LDL modification under in vivo lipid peroxidation conditions.

Co-expression of ICAM-1, VCAM-1, ELAM-1 and Hsp60 in human arterial and venous endothelial cells in response to cytokines and oxidized low-density lipoproteins.

T-cells and monocytes are the first cells infiltrating the arterial intima during the early stages of atherogenesis. Recently our laboratory has provided evidence that T-cells isolated from atherosclerotic intima reacts against heat shock protein 60 (Hsp60). Transmigration of activated T-cells into the intima is mediated by adhesion molecules (ICAM-1; VCAM-1; ELAM-1) expressed on activated endothelial cells. Here we studied the potential of cytokines (TNF-alpha, IFN-gamma, IL-1). Escherichia coli lipopolysaccharide (LPS), native and oxidized low-density lipoprotein (LDL; oxLDL) and high temperature to induce adhesion molecules as well as Hsp60 and Hsp70 expression in human endothelial cells (EC). On Northern blots, a strong signal for ICAM-1, VCAM-1 and ELAM-1 was detected after 4 h, which thereafter declined, but did not reach the basal level of untreated control cells. Heat shock induced the expression of Hsp60 and Hsp70 but not of adhesion molecules. EC were cultivated in serum-free medium, which led to the expression of adhesion molecule transcripts. Addition of LDL or oxLDL to these ECs did not alter the expression of these transcripts. The production of adhesion molecule proteins was analysed by flow cytometry. In human venous endothelial cells (HVEC) and human arterial endothelial cells (HAEC) ICAM-1 and VCAM-1 production was permanently highly induced, whereas the high level of ELAM-1 production at 4 h disappeared after 24 h. Furthermore, only HAEC, but not HVEC, produced ICAM-1, VCAM-1 and ELAM-1 after stress by moderately and highly oxLDL. LDL and oxLDL did not induce the production of Hsp60 and Hsp70. The present study demonstrates the co-expression of Hsp60 and adhesion molecules in arterial and venous EC in response to cytokine and LPS exposure, and that oxLDL is an efficient inducer of adhesion molecules in arterial EC and not in venous EC. These features provide the prerequisites for a cellular immune reaction against Hsp60 expressed by stressed EC in the initial stages of atherosclerosis.

Immunostaining of human autopsy aortas with antibodies to modified apolipoprotein B and apoprotein(a).

A systematic immunohistochemical study of different stages of atherosclerosis in human aortas was performed using several antibodies. Because oxidation of lipoproteins could be a key event in atherogenesis, an antibody against apolipoprotein B (apoB) from low-density lipoprotein (LDL) modified with the lipid peroxidation-specific aldehyde, 4-hydroxynonenal (4-HNE) (anti-4-HNE-apoB), was raised in rabbits. This antibody recognizing 4-HNE protein adducts was used in concert with an antibody to apo(a) from lipoprotein(a), considered also potentially atherogenic, as well as with an antibody and a monoclonal antibody (mAb) to apoB. Autopsy material from 12 corpses was investigated. The immunohistochemical investigation by the alkaline-phosphatase technique included control specimens regarding postmortem artifacts by autolysis and oxidation. The results from six specimens from five corpses are presented. A positive staining with the antibody to apoB but not with anti-4-HNE-apoB was seen in the normal intima. The thickened intima of early, transitional, and advanced atherosclerotic lesions and atheromata showed a predominantly extracellular staining with all antibodies and the applied mAb. To test the specificity of the staining, antibodies preadsorbed by the appropriate antigens and nonimmune sera were used, giving negative results. These findings indicated a colocalization of epitopes derived from lipid peroxidation of polyunsaturated fatty acids and epitopes specific for apoB and apo(a) during atherogenesis in humans.

Lipoprotein(a) in ischemic cerebrovascular disease A new approach to the assessment of risk for stroke

We estimated the serum levels of lipoprotein(a) [Lp(a)] of 87 patients suffering from ischemic cerebrovascular disease (ICVD) (50 men, 37 women; mean age, 66.7 f 10.9 years; median, 66) and of 66 healthy controls (33 men, 33 women; mean age, 62.2 k 11.1; median, 63). The Lp(a) serum levels of the ICVD group (mean, 20.5 k 23 mg/dl; median, 9.5) were significantly elevated compared with those of controls (mean, 14.2 k 23.1 mg/dl; median, 5). In a smaller submouu, limiting the age range to 30 to 60 vears. I. the difference in the Lp(a) serum levels between the ICVD patients and the controls was even highly significant (p < 0.001).We estimated the serum levels of lipoprotein(a) [Lp(a)] of 87 patients suffering from ischemic cerebrovascular disease (ICVD) (50 men, 37 women; mean age, 66.7 +/- 10.9 years; median, 66) and of 66 healthy controls (33 men, 33 women; mean age, 62.2 +/- 11.1; median, 63). The Lp(a) serum levels of the ICVD group (mean, 20.5 +/- 23 mg/dl; median, 9.5) were significantly elevated compared with those of controls (mean, 14.2 +/- 23.1 mg/dl; median, 5). In a smaller subgroup, limiting the age range to 30 to 60 years, the difference in the Lp(a) serum levels between the ICVD patients and the controls was even highly significant (p less than 0.001).

Activation of EGF receptor by oxidized LDL

Oxidized low density lipoproteins (oxLDL) are thought to play a major role in atherosclerosis. OxLDL exhibit a wide variety of biological effects resulting from their ability to interfere with intracellular signaling. The cellular targets and primary signaling events of oxLDL are unknown. We report that oxLDL elicit, in intact cells, tyrosine phosphorylation of the epithelial growth factor receptor (EGFR) and activation of its signaling pathway. This activation triggered by oxLDL was associated with derivatization of reactive amino groups of EGFR and was mimicked by 4‐hydroxynonenal (4‐HNE, a major lipid peroxidation product of oxLDL). Immunopurified EGFR was derivatized and activated in vitro by oxLDL lipid extracts and 4‐HNE, thus indicating that 1) EGFR may be a primary target of oxidized lipids and 2) EGFR derivatization may be associated with activation. The reported data suggest that EGFR acts as a sensor for oxidized lipids. We therefore propose a novel concept of the mechanism by which oxidized lipids (contained in oxLDL or more generally produced during oxidative stress) are able to activate receptor tyrosine kinase and subsequent signaling pathways, resulting finally in a gain of function.—Suc, Isabelle, Meilhac, Olivier, Lajoie‐MAZENC, Isabelle, Vandaele, Jean, Jurgens, GüNTHER, Salvayre, Robert, NèGRE‐SALVAYRE, Anne Activation of EGF receptor by oxidized LDL. FASEB J. 12, 665–671 (1998)

A dominant role of lipoprotein(a) in the investigation and evaluation of parameters indicating the development of cervical atherosclerosis

The correlation of serum levels of lipoprotein (a) [Lp(a)] with the progression of cervical atherosclerosis was investigated and compared with the common risk factors. The carotid arteries of 100 subjects were examined by direct bi-directional Doppler ultrasonic imaging. A highly significant elevation of the mean values of Lp(a) in group 1 (P1, with smooth surface plaques) and in group 2 (P2, with exulcerations) vs the control (P0, with no detectable plaques) was established. Low density lipoprotein cholesterol (LDL-C) was highly significantly elevated in P1, but only significantly higher in P2. Total cholesterol (TC) was significantly higher in P1 and highly significantly elevated in P2. Diabetes was also found to be significantly associated with atherosclerotic plaque formation, in contrast to triglycerides (TG), high density lipoprotein cholesterol (HDL-C) and its ratio to TC, hypertension and cigarette smoking. In a smaller collective of 30 patients--40-60 years old--being equally divided into 3 groups (p0, p1, p2), Lp(a) showed again to be the most significant parameter. LDL-C, TC and its ratio to HDL-C were highly significantly altered in subgroup p1 and significantly altered in subgroup p2. In this selection there were 12 patients with and 18 without cerebral infarction (CI). The difference of the medians of Lp(a) serum levels between these 2 groups was also found to be highly significant.

Oxidized LDLs alter the activity of the ubiquitin-proteasome pathway: potential role in oxidized LDL-induced apoptosis

Oxidized low‐density lipoproteins (ox‐LDL) play a role in the genesis of atherosclerosis. OxLDL are able to induce apoptosis of vascular cells, which is potentially involved in the formation of the necrotic center of atherosclerotic lesions, plaque rupture, and subsequent thrombotic events. Because oxLDL may induce structural modifications of cell protein and altered proteins may impair cell viability, the present work aimed to evaluate the extent of protein alterations, the degradation of modified proteins through the ubiquitin‐proteasome system (a major degradative pathway for altered and oxidatively modified proteins) and their role during apoptosis induced by oxLDL. This paper reports the following: 1) oxLDL induce derivatization of cell proteins by 4‐hydroxynonenal (4‐HNE) and ubiquiti‐nation. 2) Toxic concentrations of oxLDL elicit a biphasic effect on proteasome activity. An early and transient activation of endogenous proteolysis is followed rapidly by a subsequent decay (resulting probably from the 26S proteasome inhibition) and followed later by the inhibition of the 20S protea‐some (as assessed by inhibition of sLLVY‐MCA hydrolysis). 3) Specific inhibitors of proteasome (lac‐tacystin and proteasome inhibitor I) potentiated considerably the toxicity of oxLDL (nontoxic doses of oxLDL became severely toxic). The defect of the ubiquitination pathway (in temperature‐sensitive mutants) also potentiated the toxicity of oxLDL. This suggests that the ubiquitin‐proteasome pathway plays a role in the cellular defenses against oxLDL‐in‐duced toxicity. 4) Dinitrophenylhydrazine (DNPH), an aldehyde reagent, prevented both the oxLDL‐induced derivatization of cell proteins and subsequent cytotoxicity. Altogether, the reported data suggest that both derivatization of cell proteins (by 4‐HNE and other oxidized lipids) and inhibition of the proteasome pathway are involved in the mechanism of oxLDL‐induced apoptosis.—Vieira, O., Escargueil‐Blanc, I., Jürgens, G., Borner, C., Almeida, L., Salvayre, R., Nègre‐Salvayre, A. Oxidized LDL alter the activity of the ubiquitin‐proteasome pathway: potential role in oxidized LDL‐induced apoptosis. FASEB J. 14, 532–542 (2000)

Generation, Characterization, and Histochemical Application of Monoclonal Antibodies Selectively Recognizing Oxidatively Modified ApoB-Containing Serum Lipoproteins

To investigate either the role oxidized LDL plays in atherosclerosis or structural changes on the surface of oxidized LDL, monoclonal antibodies (mAbs) are an important tool. After immunizing mice with Cu(2+)-oxidized LDL (oxLDL) and fusion of splenocytes, hybridoma supernatants were screened and cloned. Two mAbs, OB/04 and OB/09 (IgG and IgM), were further characterized. In solid-phase fluorescence immunoassays and Western blot analysis both mAbs reacted with oxLDL, LDL oxidized by a free radical-generating azo compound, or oxVLDL but not with native LDL, acetylated LDL, oxHDL3, azo-oxidized HDL3, or HDL3 modified with malondialdehyde (MDA). In competitive immunoassays with LDL modified by oxidized fatty acid-derived aldehydes, mAb OB/09 strongly reacted with MDA-LDL or MDA-VLDL and LDL modified with 4-hydroxyhexenal followed by 4-hydroxynonenal but not with 4-hydroxyoctenal or hepta-2,4-dienal. mAb OB/04 had a weak affinity for LDL after modification with these aldehydes except for MDA-LDL. LDL modified with arachidonic acid oxidation products (AAOPs) was also recognized by this mAb. However, albumin modified either by the aldehydes applied or by AAOPs did not react with either mAb. Thus, the data indicate that each of the mAbs recognizes a different epitope that is expressed only on apoB-containing lipoproteins upon oxidative modification. An immunostaining with mAb OB/04 was obtained in areas rich in macrophages and in connective tissue of a human atherosclerotic lesion.