- Email: [email protected]
Atherogenic lipoproteins, oxidative stress, and cell death
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 Wu¨rzburg, Wu¨rzburg, Germany
Atherogenic lipoproteins, oxidative stress, and cell death. Background. Glomerulosclerosis and atherosclerosis are chronic inflammatory processes that may be influenced by oxidized lipoproteins, oxidized low-density lipoproteins (oxLDL), and oxidized lipoprotein(a) [oxLp(a)]. We hypothesize that these lipoproteins contribute to the development of glomerulosclerosis and atherosclerosis through the induction of oxidative stress, which influences cell viability. We therefore determined the impact of oxLDL and oxLp(a) on O22 formation and on necrotic and apoptotic cell death in vascular and glomerular cells. Methods. The impact of human LDL and Lp(a) (oxidized with CuSO4) on O22 formation (detected with a chemiluminescence method), apoptosis, and necrosis (determined with the annexin assay) was studied in cultured human umbilical vein endothelial cells (ECs) and in cultured human mesangial cells (MCs). Results. O22 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 in ECs). OxLDL and oxLp(a) both significantly and dosedependently increased the rate of apoptotic cell death in ECs and in MCs, with oxLp(a) being the more potent stimulus that also caused necrosis. The induction of apoptosis by oxLDL and oxLp(a) in ECs and MCs was enhanced by inhibition of the endogenous superoxide dismutase (SOD) with diethyldithio-carbamate and was blunted by the antioxidants N-acetylcysteine, vitamin C 1 E, SOD, and catalase, suggesting that oxidative stress was the stimulus for apoptosis. Conclusions. These data suggest that oxLDL and oxLp(a) contribute to inflammation by stimulating O22 formation, leading to apoptotic cell death in the vascular wall and in the glomerulus. The oxidized lipoproteins may thereby influence the pathogenesis of atherosclerosis and glomerulosclerosis.
Glomerulosclerosis and atherosclerosis have some pathogenic pathways in common [1], and the underlying causes of the diseases are chronic inflammatory processes [2, 3]. The inflammation is maintained by a variety of pathogenic factors, among them the lipoproteins LDL and Lp(a) [4–6]. These lipoproteins increase their proinflammatory potential after oxidation [7] and accumulate in an oxidized form in the vascular wall [8] and in the glomerulus [9]. The arteries of hypercholesterolemic rabKey words: apoptosis, glomerulosclerosis, lipoprotein(a), low-density lipoproteins, pathogenesis.
1999 by the International Society of Nephrology
bits produce relatively high amounts of O22 [10], and oxygen radicals are also involved in the pathogenesis of glomerular diseases [11]. We therefore hypothesize that a major contribution of oxidized lipoproteins to the development of glomerulosclerosis and atherosclerosis consists of the induction of oxidative stress. Oxidative stress attenuates cell viability (apoptotic and necrotic cell death) [12] and, in turn, may influence the development of glomerulosclerosis [13] and atherosclerosis. Indeed, vascular regions that are prone to the development of atherosclerotic lesions are characterized by increased cell turnover during the early phase of the disease [14]. Apoptosis has also been found in human and animal atherosclerotic lesions [15, 16]. It was therefore the aim of this study to determine the impact of oxidized lowdensity lipoproteins (oxLDL), and oxidized lipoprotein(a) [oxLp(a)] on O22 formation and on necrotic and apoptotic cell death in vascular and glomerular cells. METHODS Human LDL and Lp(a) were isolated by density gradient ultracentrifugation from fresh human plasma and, in case of Lp(a), by additional lysine-sepharose 4B chromatography as described recently [17]. Lp(a) and LDL were oxidized by incubation with Cu11, and the degree of oxidation was analyzed by agarose gel electrophoresis as described [17]. Human umbilical vein endothelial cells (ECs) were purchased from Cell Systems/Clonetics (Walkersville, MD, USA) and were cultured in endothelial basal medium supplemented with hydrocortisone (1 mg/ml), bovine brain extract (12 mg/ml), gentamicin (50 mg/ml), amphotericin B (50 ng/ml), epidermal growth factor (10 ng/ml), and 10% fetal calf serum until the fourth passage. Mesangial cells (MCs) were established from cortex of human kidneys. Glomeruli, isolated by a sieving technique and cultured in vitro, gave rise to primary MC cultures. After characterization with antibodies, the cells were used for experiments in the fifth passage. Apoptosis was detected and distinguished from necrosis after 18 hours incubation with the lipoproteins by staining with Annexin-V-FLUOS. One 3 106 cells were
S-62
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Galle et al: Lipoproteins and apoptosis
Fig. 1. Lipoproteins stimulate O22 formation in cultured endothelial cells. Time course of the O22 formation in terms of chemiluminescence of lucigenin (counts per sec/mg protein) in untreated cultured ECs (control) and in ECs treated with 5 and 10 mg/ml oxLDL (left panel) or treated with 1, 2.5, and 5 mg/ml oxLp(a) (right panel). Incubations were carried out in the presence of diethyl-dithio-carbamate, an inhibitor of the endogenous superoxide dismutase (SOD). The lipoproteins significantly stimulated O22 formation of the endothelial cells. Data are means 6 se of 8 to 10 independent experiments.
washed with phosphate-buffered saline, removed from the culture dish with trypsin/ethylenediaminetetraacetic acid, centrifuged at 200 3 g, and resuspended in 150 ml 10 mm HEPES/NaOH, pH 7.4, 140 mm NaCl, 5 mm CaCl2 containing 1 mg/ml propidium iodid, and 20 ml/ml Annexin-V-FLUOS. After a 15-minute incubation period at room temperature, 500 ml cell culture medium were added, and the cells were analyzed by flow cytometry (FACScan; Becton Dickinson, Franklin Lakes, NJ, USA). O22 formation from ECs and MCs was detected by a chemiluminescence assay with lucigenin that reacts specifically with O22, resulting in the release of photons. The detection of chemiluminescence with lucigenin was carried out as described recently [17] in a scintillation counter with a single photomultiplier tube (LUMAT LB 9501/16; Berthold-Instruments). RESULTS Oxidized low-density lipoprotein and oxidized lipoprotein(a) induce oxidative stress Native lipoproteins had no effect on O22 production of ECs or MCs. However, the treatment with oxLDL or oxLp(a) significantly increased the O22 concentration in both systems, with oxLp(a) being the more potent stimulus for O22 generation. Figure 1 shows the increase of O22 formation in ECs after treatment with oxLDL or oxLp(a). Principally identical effects were observed in MCs, again with oxLp(a) being the more potent stimulus for O22 generation (data not shown). Oxidized low-density lipoprotein and oxidized lipoprotein(a) induce apoptosis Apoptosis in ECs and MCs was detected by DNA fragmentation or by an Annexin assay, which allowed us to distinguish apoptosis from necrosis by propidium
iodid staining. Incubation of ECs or MCs with oxLDL (30 to 200 mg/ml) or oxLp(a) (30 to 200 mg/ml) resulted in a dose-dependent induction of apoptosis, whereas nLDL had no and nLp(a) had little influence on cell survival. Figure 2 depicts the effects of native and oxidized LDL and Lp(a) on apoptosis and necrosis in MCs. In good correlation to the effects of the lipoproteins on oxidative stress in ECs and in MCs, comparable effects on apoptosis were observed also in ECs (data not shown). Apoptosis is mediated by reactive oxygen In order to investigate whether oxidative stress might be involved in the induction of apoptosis in ECs and MCs, we incubated the cells with antioxidants: In MCs, coincubation during the stimulation with lipoproteins for 18 hours with N-acetyl-L-cystein (NAC) (200 mm) as well 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 the O22-catabolizing enzymes superoxide dismutase (SOD) (0.1 mm) or catalase (100 U/ml) on the rate of apoptosis. During the 18-hour incubation period, apoptosis in ECs induced by oxLDL was inhibited by SOD and by catalase by 82 and 81%, respectively, and apoptosis induced by oxLp(a) was inhibited by SOD and by catalase by 63 and by 94%, respectively. When given in combination, SOD and catalase decreased the apoptosis rate induced by oxLDL or by oxLp(a) below control levels. Thus, stimulation of apoptosis by oxLDL and oxLp(a) seems to be mediated by oxidative stress. CONCLUSIONS OxLDL and oxLp(a) induce O22 formation and apoptosis in ECs and in MCs, with oxLp(a) being the more potent stimulus. Antioxidants prevent the induction of apoptosis, underlining the role of oxidative stress in lipo-
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Galle et al: Lipoproteins and apoptosis
Fig. 2. Lipoproteins induce necrotic and apoptotic cell death in cultured mesangial cells. Dose-response curves of the effect of low-density lipoprotein (LDL; upper panel) and Lp(a) (lower panel) on the survival of primary mesangial cell (MC) cultures. MCs were treated with native or oxidized lipoprotein for 18 hours. Apoptosis was detected using an Annexin assay. Necrosis was detected by propidium iodid staining. OxLDL as well as oxLp(a) dose dependently caused apoptosis. Data are means 6 se of three independent experiments.
protein-mediated cell death. Thereby, oxidized lipoproteins may influence the pathogenesis of atherosclerosis and glomerulosclerosis. ACKNOWLEDGMENTS This study was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG Ga 431/2-1). Reprint requests to Jan Galle, M.D., Department of Medicine, Division of Nephrology, University of Hospital Wu¨rzburg, Joseph-Schneider-Str. 2, D-97080 Wu¨rzburg, Germany. E-mail: [email protected]
REFERENCES 1. Magil AB, Frohlich JJ, Innis SM, Steinbrecher UP: Oxidized low-density lipoprotein in experimental focal glomerulosclerosis. Kidney Int 43:1243–1250, 1993 2. Holvoet P, Collen D: Thrombosis and atherosclerosis. Curr Opin Lipidol 8:320–328, 1997 3. Sterzel RB, Schulze-Lohoff E, Marx M: Cytokines and mesangial cells. Kidney Int 43(Suppl 39):S26–S31, 1993 4. Wheeler DC, Chana RS: Interactions between lipoproteins, glomerular cells and matrix. Miner Electrolyte Metab 19:149–164, 1993 5. Ding GH, Van Goor H, Ricardo SD, Orlowski N, Diamond JR: Oxidized LDL stimulates the expression of TGF-b and fibronectin in human glomerular epithelial cells. Kidney Int 51:147–154, 1997 6. Scanu AM, Edelstein C: Learning about the structure and biology
Galle et al: Lipoproteins and apoptosis
7. 8.
9. 10.
11. 12.
of human lipoprotein [a] through dissection by enzymes of the elastase family: Facts and speculations. J Lipid Res 38:2193–2206, 1997 Berliner JA, Heinecke JW: The role of oxidized lipoproteins in atherogenesis. Free Radic Biol Med 20:707–727, 1996 Yla¨-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witzum JL, Steinberg D: Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest 84:1086– 1095, 1989 Lee HS, Kim YS: Identification of oxidized low density lipoprotein in human renal biopsies. Kidney Int 54:848–856, 1998 Ohara Y, Peterson TE, Harrison DG: Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest 91:2546–2551, 1993 Klahr S: Oxygen radicals and renal diseases. Miner Electrolyte Metab 23:140–143, 1997 Hug H, Strand S, Grambihler A, Galle J, Hack V, Stremmel
13. 14. 15.
16. 17.
S-65
W, Krammer PH, Galle PR: Communication: Reactive oxygen intermediates are involved in the induction of CD95 ligand mRNA expression by cytostatic drugs in hepatoma cells. J Biol Chem 272:28191–28193, 1997 Sugiyama H, Kashihara N, Makino H, Yamasaki Y, Ota Z: Apoptosis in glomerular sclerosis. Kidney Int 49:103–111, 1996 Caplan BA, Schwartz CJ: Increased endothelial cell turnover in areas of in vivo Evans blue uptake in the pig aorta. Atherosclerosis 17:401–417, 1973 Bjo¨rkerud S, Bjo¨rkerud B: Apoptosis is abundant in human atherosclerotic lesions, especially in inflammatory cells (macrophages and T cells), and may contribute to the accumulation of gruel and plaque instability. Am J Pathol 149:367–380, 1996 Han DKM, Haudenschild CC, Hong MK, Tinkle BT, Leon MB, Liau G: Evidence for apoptosis in human atherogenesis and in a rat vascular injury model. Am J Pathol 147:267–277, 1995 Galle J, Heinloth A, Schwedler S, Wanner C: Effect of HDL and atherogenic lipoproteins on formation of O22 and renin release in juxtaglomerular cells. Kidney Int 51:253–260, 1997
Atherogenic lipoproteins, oxidative stress, and cell death JAN GALLE, KATHRIN HEERMEIER, and CHRISTOPH WANNER Department of Medicine, Division of Nephrology, University Hospital of Wu¨rzburg, Wu¨rzburg, Germany
Atherogenic lipoproteins, oxidative stress, and cell death. Background. Glomerulosclerosis and atherosclerosis are chronic inflammatory processes that may be influenced by oxidized lipoproteins, oxidized low-density lipoproteins (oxLDL), and oxidized lipoprotein(a) [oxLp(a)]. We hypothesize that these lipoproteins contribute to the development of glomerulosclerosis and atherosclerosis through the induction of oxidative stress, which influences cell viability. We therefore determined the impact of oxLDL and oxLp(a) on O22 formation and on necrotic and apoptotic cell death in vascular and glomerular cells. Methods. The impact of human LDL and Lp(a) (oxidized with CuSO4) on O22 formation (detected with a chemiluminescence method), apoptosis, and necrosis (determined with the annexin assay) was studied in cultured human umbilical vein endothelial cells (ECs) and in cultured human mesangial cells (MCs). Results. O22 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 in ECs). OxLDL and oxLp(a) both significantly and dosedependently increased the rate of apoptotic cell death in ECs and in MCs, with oxLp(a) being the more potent stimulus that also caused necrosis. The induction of apoptosis by oxLDL and oxLp(a) in ECs and MCs was enhanced by inhibition of the endogenous superoxide dismutase (SOD) with diethyldithio-carbamate and was blunted by the antioxidants N-acetylcysteine, vitamin C 1 E, SOD, and catalase, suggesting that oxidative stress was the stimulus for apoptosis. Conclusions. These data suggest that oxLDL and oxLp(a) contribute to inflammation by stimulating O22 formation, leading to apoptotic cell death in the vascular wall and in the glomerulus. The oxidized lipoproteins may thereby influence the pathogenesis of atherosclerosis and glomerulosclerosis.
Glomerulosclerosis and atherosclerosis have some pathogenic pathways in common [1], and the underlying causes of the diseases are chronic inflammatory processes [2, 3]. The inflammation is maintained by a variety of pathogenic factors, among them the lipoproteins LDL and Lp(a) [4–6]. These lipoproteins increase their proinflammatory potential after oxidation [7] and accumulate in an oxidized form in the vascular wall [8] and in the glomerulus [9]. The arteries of hypercholesterolemic rabKey words: apoptosis, glomerulosclerosis, lipoprotein(a), low-density lipoproteins, pathogenesis.
1999 by the International Society of Nephrology
bits produce relatively high amounts of O22 [10], and oxygen radicals are also involved in the pathogenesis of glomerular diseases [11]. We therefore hypothesize that a major contribution of oxidized lipoproteins to the development of glomerulosclerosis and atherosclerosis consists of the induction of oxidative stress. Oxidative stress attenuates cell viability (apoptotic and necrotic cell death) [12] and, in turn, may influence the development of glomerulosclerosis [13] and atherosclerosis. Indeed, vascular regions that are prone to the development of atherosclerotic lesions are characterized by increased cell turnover during the early phase of the disease [14]. Apoptosis has also been found in human and animal atherosclerotic lesions [15, 16]. It was therefore the aim of this study to determine the impact of oxidized lowdensity lipoproteins (oxLDL), and oxidized lipoprotein(a) [oxLp(a)] on O22 formation and on necrotic and apoptotic cell death in vascular and glomerular cells. METHODS Human LDL and Lp(a) were isolated by density gradient ultracentrifugation from fresh human plasma and, in case of Lp(a), by additional lysine-sepharose 4B chromatography as described recently [17]. Lp(a) and LDL were oxidized by incubation with Cu11, and the degree of oxidation was analyzed by agarose gel electrophoresis as described [17]. Human umbilical vein endothelial cells (ECs) were purchased from Cell Systems/Clonetics (Walkersville, MD, USA) and were cultured in endothelial basal medium supplemented with hydrocortisone (1 mg/ml), bovine brain extract (12 mg/ml), gentamicin (50 mg/ml), amphotericin B (50 ng/ml), epidermal growth factor (10 ng/ml), and 10% fetal calf serum until the fourth passage. Mesangial cells (MCs) were established from cortex of human kidneys. Glomeruli, isolated by a sieving technique and cultured in vitro, gave rise to primary MC cultures. After characterization with antibodies, the cells were used for experiments in the fifth passage. Apoptosis was detected and distinguished from necrosis after 18 hours incubation with the lipoproteins by staining with Annexin-V-FLUOS. One 3 106 cells were
S-62
S-63
Galle et al: Lipoproteins and apoptosis
Fig. 1. Lipoproteins stimulate O22 formation in cultured endothelial cells. Time course of the O22 formation in terms of chemiluminescence of lucigenin (counts per sec/mg protein) in untreated cultured ECs (control) and in ECs treated with 5 and 10 mg/ml oxLDL (left panel) or treated with 1, 2.5, and 5 mg/ml oxLp(a) (right panel). Incubations were carried out in the presence of diethyl-dithio-carbamate, an inhibitor of the endogenous superoxide dismutase (SOD). The lipoproteins significantly stimulated O22 formation of the endothelial cells. Data are means 6 se of 8 to 10 independent experiments.
washed with phosphate-buffered saline, removed from the culture dish with trypsin/ethylenediaminetetraacetic acid, centrifuged at 200 3 g, and resuspended in 150 ml 10 mm HEPES/NaOH, pH 7.4, 140 mm NaCl, 5 mm CaCl2 containing 1 mg/ml propidium iodid, and 20 ml/ml Annexin-V-FLUOS. After a 15-minute incubation period at room temperature, 500 ml cell culture medium were added, and the cells were analyzed by flow cytometry (FACScan; Becton Dickinson, Franklin Lakes, NJ, USA). O22 formation from ECs and MCs was detected by a chemiluminescence assay with lucigenin that reacts specifically with O22, resulting in the release of photons. The detection of chemiluminescence with lucigenin was carried out as described recently [17] in a scintillation counter with a single photomultiplier tube (LUMAT LB 9501/16; Berthold-Instruments). RESULTS Oxidized low-density lipoprotein and oxidized lipoprotein(a) induce oxidative stress Native lipoproteins had no effect on O22 production of ECs or MCs. However, the treatment with oxLDL or oxLp(a) significantly increased the O22 concentration in both systems, with oxLp(a) being the more potent stimulus for O22 generation. Figure 1 shows the increase of O22 formation in ECs after treatment with oxLDL or oxLp(a). Principally identical effects were observed in MCs, again with oxLp(a) being the more potent stimulus for O22 generation (data not shown). Oxidized low-density lipoprotein and oxidized lipoprotein(a) induce apoptosis Apoptosis in ECs and MCs was detected by DNA fragmentation or by an Annexin assay, which allowed us to distinguish apoptosis from necrosis by propidium
iodid staining. Incubation of ECs or MCs with oxLDL (30 to 200 mg/ml) or oxLp(a) (30 to 200 mg/ml) resulted in a dose-dependent induction of apoptosis, whereas nLDL had no and nLp(a) had little influence on cell survival. Figure 2 depicts the effects of native and oxidized LDL and Lp(a) on apoptosis and necrosis in MCs. In good correlation to the effects of the lipoproteins on oxidative stress in ECs and in MCs, comparable effects on apoptosis were observed also in ECs (data not shown). Apoptosis is mediated by reactive oxygen In order to investigate whether oxidative stress might be involved in the induction of apoptosis in ECs and MCs, we incubated the cells with antioxidants: In MCs, coincubation during the stimulation with lipoproteins for 18 hours with N-acetyl-L-cystein (NAC) (200 mm) as well 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 the O22-catabolizing enzymes superoxide dismutase (SOD) (0.1 mm) or catalase (100 U/ml) on the rate of apoptosis. During the 18-hour incubation period, apoptosis in ECs induced by oxLDL was inhibited by SOD and by catalase by 82 and 81%, respectively, and apoptosis induced by oxLp(a) was inhibited by SOD and by catalase by 63 and by 94%, respectively. When given in combination, SOD and catalase decreased the apoptosis rate induced by oxLDL or by oxLp(a) below control levels. Thus, stimulation of apoptosis by oxLDL and oxLp(a) seems to be mediated by oxidative stress. CONCLUSIONS OxLDL and oxLp(a) induce O22 formation and apoptosis in ECs and in MCs, with oxLp(a) being the more potent stimulus. Antioxidants prevent the induction of apoptosis, underlining the role of oxidative stress in lipo-
S-64
Galle et al: Lipoproteins and apoptosis
Fig. 2. Lipoproteins induce necrotic and apoptotic cell death in cultured mesangial cells. Dose-response curves of the effect of low-density lipoprotein (LDL; upper panel) and Lp(a) (lower panel) on the survival of primary mesangial cell (MC) cultures. MCs were treated with native or oxidized lipoprotein for 18 hours. Apoptosis was detected using an Annexin assay. Necrosis was detected by propidium iodid staining. OxLDL as well as oxLp(a) dose dependently caused apoptosis. Data are means 6 se of three independent experiments.
protein-mediated cell death. Thereby, oxidized lipoproteins may influence the pathogenesis of atherosclerosis and glomerulosclerosis. ACKNOWLEDGMENTS This study was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG Ga 431/2-1). Reprint requests to Jan Galle, M.D., Department of Medicine, Division of Nephrology, University of Hospital Wu¨rzburg, Joseph-Schneider-Str. 2, D-97080 Wu¨rzburg, Germany. E-mail: [email protected]
REFERENCES 1. Magil AB, Frohlich JJ, Innis SM, Steinbrecher UP: Oxidized low-density lipoprotein in experimental focal glomerulosclerosis. Kidney Int 43:1243–1250, 1993 2. Holvoet P, Collen D: Thrombosis and atherosclerosis. Curr Opin Lipidol 8:320–328, 1997 3. Sterzel RB, Schulze-Lohoff E, Marx M: Cytokines and mesangial cells. Kidney Int 43(Suppl 39):S26–S31, 1993 4. Wheeler DC, Chana RS: Interactions between lipoproteins, glomerular cells and matrix. Miner Electrolyte Metab 19:149–164, 1993 5. Ding GH, Van Goor H, Ricardo SD, Orlowski N, Diamond JR: Oxidized LDL stimulates the expression of TGF-b and fibronectin in human glomerular epithelial cells. Kidney Int 51:147–154, 1997 6. Scanu AM, Edelstein C: Learning about the structure and biology
Galle et al: Lipoproteins and apoptosis
7. 8.
9. 10.
11. 12.
of human lipoprotein [a] through dissection by enzymes of the elastase family: Facts and speculations. J Lipid Res 38:2193–2206, 1997 Berliner JA, Heinecke JW: The role of oxidized lipoproteins in atherogenesis. Free Radic Biol Med 20:707–727, 1996 Yla¨-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witzum JL, Steinberg D: Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest 84:1086– 1095, 1989 Lee HS, Kim YS: Identification of oxidized low density lipoprotein in human renal biopsies. Kidney Int 54:848–856, 1998 Ohara Y, Peterson TE, Harrison DG: Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest 91:2546–2551, 1993 Klahr S: Oxygen radicals and renal diseases. Miner Electrolyte Metab 23:140–143, 1997 Hug H, Strand S, Grambihler A, Galle J, Hack V, Stremmel
13. 14. 15.
16. 17.
S-65
W, Krammer PH, Galle PR: Communication: Reactive oxygen intermediates are involved in the induction of CD95 ligand mRNA expression by cytostatic drugs in hepatoma cells. J Biol Chem 272:28191–28193, 1997 Sugiyama H, Kashihara N, Makino H, Yamasaki Y, Ota Z: Apoptosis in glomerular sclerosis. Kidney Int 49:103–111, 1996 Caplan BA, Schwartz CJ: Increased endothelial cell turnover in areas of in vivo Evans blue uptake in the pig aorta. Atherosclerosis 17:401–417, 1973 Bjo¨rkerud S, Bjo¨rkerud B: Apoptosis is abundant in human atherosclerotic lesions, especially in inflammatory cells (macrophages and T cells), and may contribute to the accumulation of gruel and plaque instability. Am J Pathol 149:367–380, 1996 Han DKM, Haudenschild CC, Hong MK, Tinkle BT, Leon MB, Liau G: Evidence for apoptosis in human atherogenesis and in a rat vascular injury model. Am J Pathol 147:267–277, 1995 Galle J, Heinloth A, Schwedler S, Wanner C: Effect of HDL and atherogenic lipoproteins on formation of O22 and renin release in juxtaglomerular cells. Kidney Int 51:253–260, 1997