Kidney International, Vol. 56, Suppl. 71 (1999), pp. S-62–S-65

Atherogenic lipoproteins, oxidative stress, and cell death

JAN GALLE, KATHRIN HEERMEIER, and CHRISTOPH WANNER

Department of Medicine, Division of Nephrology, University Hospital of Wurzburg, Wurzburg, Germany

Atherogenic lipoproteins, oxidative stress, and cell death. bits produce relatively high amounts of O22 [10], and

Background. Glomerulosclerosis and atherosclerosis are oxygen radicals are also involved in the pathogenesis ofchronic inflammatory processes that may be influenced by oxi- glomerular diseases [11]. We therefore hypothesize thatdized lipoproteins, oxidized low-density lipoproteins (oxLDL),

a major contribution of oxidized lipoproteins to the de-and oxidized lipoprotein(a) [oxLp(a)]. We hypothesize thatvelopment of glomerulosclerosis and atherosclerosisthese lipoproteins contribute to the development of glomerulo-

sclerosis and atherosclerosis through the induction of oxidative consists of the induction of oxidative stress. Oxidativestress, which influences cell viability. We therefore determined stress attenuates cell viability (apoptotic and necroticthe impact of oxLDL and oxLp(a) on O2

2 formation and on cell death) [12] and, in turn, may influence the develop-necrotic and apoptotic cell death in vascular and glomerularment of glomerulosclerosis [13] and atherosclerosis. In-cells.deed, vascular regions that are prone to the developmentMethods. The impact of human LDL and Lp(a) (oxidized

with CuSO4) on O22 formation (detected with a chemilumines- of atherosclerotic lesions are characterized by increased

cence method), apoptosis, and necrosis (determined with the cell turnover during the early phase of the disease [14].annexin assay) was studied in cultured human umbilical vein Apoptosis has also been found in human and animalendothelial cells (ECs) and in cultured human mesangial cells

atherosclerotic lesions [15, 16]. It was therefore the aim(MCs).of this study to determine the impact of oxidized low-Results. O2

2 formation was increased by 10 mg/ml oxLDL(by factor 2.5 in ECs) and by 5 mg/ml oxLp(a) (by factor 3.5 density lipoproteins (oxLDL), and oxidized lipopro-in ECs). OxLDL and oxLp(a) both significantly and dose- tein(a) [oxLp(a)] on O2

2 formation and on necrotic anddependently increased the rate of apoptotic cell death in ECs apoptotic cell death in vascular and glomerular cells.and in MCs, with oxLp(a) being the more potent stimulus thatalso caused necrosis. The induction of apoptosis by oxLDLand oxLp(a) in ECs and MCs was enhanced by inhibition

METHODSof the endogenous superoxide dismutase (SOD) with diethyl-dithio-carbamate and was blunted by the antioxidants N-acetyl- Human LDL and Lp(a) were isolated by density gradi-cysteine, vitamin C 1 E, SOD, and catalase, suggesting that ent ultracentrifugation from fresh human plasma and, inoxidative stress was the stimulus for apoptosis. case of Lp(a), by additional lysine-sepharose 4B chroma-Conclusions. These data suggest that oxLDL and oxLp(a)

tography as described recently [17]. Lp(a) and LDL werecontribute to inflammation by stimulating O22 formation, lead-

oxidized by incubation with Cu11, and the degree ofing to apoptotic cell death in the vascular wall and in theglomerulus. The oxidized lipoproteins may thereby influence oxidation was analyzed by agarose gel electrophoresisthe pathogenesis of atherosclerosis and glomerulosclerosis. as described [17].

Human umbilical vein endothelial cells (ECs) werepurchased from Cell Systems/Clonetics (Walkersville,

Glomerulosclerosis and atherosclerosis have some MD, USA) and were cultured in endothelial basal me-pathogenic pathways in common [1], and the underlying dium supplemented with hydrocortisone (1 mg/ml), bo-causes of the diseases are chronic inflammatory processes vine brain extract (12 mg/ml), gentamicin (50 mg/ml),[2, 3]. The inflammation is maintained by a variety of amphotericin B (50 ng/ml), epidermal growth factor (10pathogenic factors, among them the lipoproteins LDL ng/ml), and 10% fetal calf serum until the fourth passage.and Lp(a) [4–6]. These lipoproteins increase their proin- Mesangial cells (MCs) were established from cortexflammatory potential after oxidation [7] and accumulate of human kidneys. Glomeruli, isolated by a sieving tech-in an oxidized form in the vascular wall [8] and in the nique and cultured in vitro, gave rise to primary MCglomerulus [9]. The arteries of hypercholesterolemic rab- cultures. After characterization with antibodies, the cells

were used for experiments in the fifth passage.Apoptosis was detected and distinguished from necro-Key words: apoptosis, glomerulosclerosis, lipoprotein(a), low-density

lipoproteins, pathogenesis. sis after 18 hours incubation with the lipoproteins bystaining with Annexin-V-FLUOS. One 3 106 cells were 1999 by the International Society of Nephrology

S-62

Galle et al: Lipoproteins and apoptosis S-63

Fig. 1. Lipoproteins stimulate O22 formation

in cultured endothelial cells. Time course ofthe O2

2 formation in terms of chemilumines-cence of lucigenin (counts per sec/mg protein)in untreated cultured ECs (control) and inECs treated with 5 and 10 mg/ml oxLDL (leftpanel) or treated with 1, 2.5, and 5 mg/mloxLp(a) (right panel). Incubations were car-ried out in the presence of diethyl-dithio-car-bamate, an inhibitor of the endogenous super-oxide dismutase (SOD). The lipoproteinssignificantly stimulated O2

2 formation of theendothelial cells. Data are means 6 se of 8to 10 independent experiments.

washed with phosphate-buffered saline, removed from iodid staining. Incubation of ECs or MCs with oxLDLthe culture dish with trypsin/ethylenediaminetetraacetic (30 to 200 mg/ml) or oxLp(a) (30 to 200 mg/ml) resulted inacid, centrifuged at 200 3 g, and resuspended in 150 a dose-dependent induction of apoptosis, whereas nLDLml 10 mm HEPES/NaOH, pH 7.4, 140 mm NaCl, 5 mm had no and nLp(a) had little influence on cell survival.CaCl2 containing 1 mg/ml propidium iodid, and 20 Figure 2 depicts the effects of native and oxidized LDLml/ml Annexin-V-FLUOS. After a 15-minute incubation and Lp(a) on apoptosis and necrosis in MCs. In goodperiod at room temperature, 500 ml cell culture medium correlation to the effects of the lipoproteins on oxidativewere added, and the cells were analyzed by flow cytome- stress in ECs and in MCs, comparable effects on apopto-try (FACScan; Becton Dickinson, Franklin Lakes, NJ, sis were observed also in ECs (data not shown).USA).

Apoptosis is mediated by reactive oxygenO22 formation from ECs and MCs was detected by

a chemiluminescence assay with lucigenin that reacts In order to investigate whether oxidative stress mightspecifically with O2

2, resulting in the release of photons. be involved in the induction of apoptosis in ECs andThe detection of chemiluminescence with lucigenin was MCs, we incubated the cells with antioxidants: In MCs,carried out as described recently [17] in a scintillation coincubation during the stimulation with lipoproteins forcounter with a single photomultiplier tube (LUMAT LB 18 hours with N-acetyl-L-cystein (NAC) (200 mm) as well9501/16; Berthold-Instruments). as with a combination of vitamins C and E (10 mm each)

prevented oxLDL induced apoptosis by 55 and 46%,respectively. In ECs, we investigated the influence of theRESULTSO2

2-catabolizing enzymes superoxide dismutase (SOD)Oxidized low-density lipoprotein and oxidized (0.1 mm) or catalase (100 U/ml) on the rate of apoptosis.lipoprotein(a) induce oxidative stress During the 18-hour incubation period, apoptosis in ECs

Native lipoproteins had no effect on O22 production induced by oxLDL was inhibited by SOD and by catalase

of ECs or MCs. However, the treatment with oxLDL or by 82 and 81%, respectively, and apoptosis induced byoxLp(a) significantly increased the O2

2 concentration oxLp(a) was inhibited by SOD and by catalase by 63in both systems, with oxLp(a) being the more potent and by 94%, respectively. When given in combination,stimulus for O2

2 generation. Figure 1 shows the increase SOD and catalase decreased the apoptosis rate inducedof O2

2 formation in ECs after treatment with oxLDL or by oxLDL or by oxLp(a) below control levels. Thus,oxLp(a). Principally identical effects were observed in stimulation of apoptosis by oxLDL and oxLp(a) seemsMCs, again with oxLp(a) being the more potent stimulus to be mediated by oxidative stress.for O2

2 generation (data not shown).

CONCLUSIONSOxidized low-density lipoprotein and oxidizedlipoprotein(a) induce apoptosis OxLDL and oxLp(a) induce O2

2 formation and apo-ptosis in ECs and in MCs, with oxLp(a) being the moreApoptosis in ECs and MCs was detected by DNApotent stimulus. Antioxidants prevent the induction offragmentation or by an Annexin assay, which allowed

us to distinguish apoptosis from necrosis by propidium apoptosis, underlining the role of oxidative stress in lipo-

Galle et al: Lipoproteins and apoptosisS-64

Fig. 2. Lipoproteins induce necrotic andapoptotic cell death in cultured mesangialcells. Dose-response curves of the effect oflow-density lipoprotein (LDL; upper panel)and Lp(a) (lower panel) on the survival ofprimary mesangial cell (MC) cultures. MCswere treated with native or oxidized lipopro-tein for 18 hours. Apoptosis was detected us-ing an Annexin assay. Necrosis was detectedby propidium iodid staining. OxLDL as well asoxLp(a) dose dependently caused apoptosis.Data are means 6 se of three independentexperiments.

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