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Oxidized Lipids and Lysosomal Pathology in Atherogenesis
Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
2000 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Macrophages take up large amount of exogenous materials such as oxidatively modified low-density lipoproteins (oxLDL) and lysosomotropic agents. OxLDL is taken up into macrophage lysosomes through receptor-mediated endocytosis, but poorly degraded, resulting in foam cell formation. Cholesterol oxidation products, major toxic components of oxLDL, are involved in foam cell formation and the initiation of atherosclerosis. The production of ROS/RNS, cytokines, and matrix proteases by macrophages and the apoptosis of arterial cells may contribute to atherosclerotic plaque development and destabilization.

We had four objectives in this study, first we study the influence of oxLDL on lysosomal membrane stability, location and activity of lysosomal enzymes, macrophage cell death, and the modulation of these cellular characteristics by high-density lipoprotein (HDL), vitantin E (vit E), and the iron chelator desferrioximine (DFO) or iron complex. Second, we examined the role of lysosomal enzymes in macrophage apoptosis induced by oxysterols, the major cytotoxic components of oxLDL. Third, we analyzed the expression of cysteine protease cathepsins B and L and studied their relationship to macrophage apoptosis in human atherosclerotic lesions. And fourth, we investigated whether lysosomal rupture and release of lysosomal enzymes could initiate apoptosis using the lysosomotropic detergent MSDH.

The results suggest that oxLDL causes lysosomal destabilization and relocation of lysos'omal enzymes as indicated by increased cytosolic NABGase, cathepsin-L, and cathepsin D, and decreased lysosomal acridine orange (AO)-induced red fluorescence. AcLDL had no cytotoxic effects on the cells and their lysosomes. HDL, vit E, and the iron chelator DFO diminished the cytotoxicity of oxLDL by decreasing lysosomal damage, while the iron complex enhanced oxLDL cytotoxicity. Macrophage apoptosis induced by ChOx and 7-oxysterols (7B-OH and 7- keto) is associated with lysosomal rupture and release of lysosomal enzymes to the cytosol. The lysosomal punctuated immune-granularity of cathepsins B and L was decreased in 7 -oxysteroltreated cells compared to control cells. Moreover, there is enhancement and dispersion of cathepsins B and L immunoreactivity throughout 7-oxysterols-treated cells and an extensive immunoreaction in the nuclei and around nuclear areas of apoptotic cells. Using MSDH, a lysosomotropic detergent, we further demonstrated that lysosomal rupture and release of lytic enzymes indeed play an initial role in macrophage apoptosis.

There is a lesion dependent eo-expression of cathepsins B and Land caspase-3 in early and advanced human atherosclerotic lesions, which is associated with macrophage apoptosis.

We conclude that macrophage cell death including apoptosis induced by oxLDL and oxysterols is associated with lysosomal rupture and relocation of lysosomal hydrolytic enzymes to the cytosol. The released enzymes may initiate ceU death by activating the caspase cascade, attacking mitochondria, or directly attacking the nuclear of the cells. The high eo-expression of cathepsins B and L with caspase-3 in apoptotic macrophages in human atheroma lesions suggests that macrophage apoptosis and related hydrolytic enzymes may play an important role in lesion development and plaque instability.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2000. , 56 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 652
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-28056Local ID: 12819ISBN: 91-7219-753-6 (print)OAI: oai:DiVA.org:liu-28056DiVA: diva2:248607
Public defence
2000-12-01, Berzeliussalen, Universitetssjukhuset, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-08-16Bibliographically approved
List of papers
1. Uptake of Oxidized LDL by Macrophages Results in Partial Lysosomal Enzyme Inactivation and Relocation
Open this publication in new window or tab >>Uptake of Oxidized LDL by Macrophages Results in Partial Lysosomal Enzyme Inactivation and Relocation
1998 (English)In: Arteriosclerosis, Thrombosis and Vascular Biology, ISSN 1079-5642, E-ISSN 1524-4636, Vol. 18, no 2, 177-84 p.Article in journal (Refereed) Published
Abstract [en]

The cytotoxicity of oxidized LDL (oxLDL) to several types of artery wall cells might contribute to atherosclerosis by causing cell death, presumably by both apoptosis and necrosis. After its uptake into macrophage lysosomes by receptor-mediated endocytosis, oxLDL is poorly degraded, resulting in ceroid-containing foam cells. We studied the influence of oxLDL on lysosomal enzyme activity and, in particular, on lysosomal membrane stability and the modulation of these cellular characteristics by HDL and vitamin E (vit-E). Unexposed cells and cells exposed to acetylated LDL (AcLDL) were used as controls. The lysosomal marker enzymes cathepsin L and N-acetyl-β-glucosaminidase (NAβGase) were biochemically assayed in J-774 cells after fractionation. Lysosomal integrity in living cells was assayed by the acridine orange (AO) relocation test. Cathepsin D was immunocytochemically demonstrated in J-774 cells and human monocyte-derived macrophages. We found that the total activities of NAβGase and cathepsin L were significantly decreased, whereas their relative cytosolic activities were enhanced, after oxLDL exposure. Labilization of the lysosomal membranes was further proven by decreased lysosomal AO uptake and relocation to the cytosol of cathepsin D, as estimated by light and electron microscopic immunocytochemistry. HDL and vit-E diminished the cytotoxicity of oxLDL by decreasing the lysosomal damage. The results indicate that endocytosed oxLDL not only partially inactivates lysosomal enzymes but also destabilizes the acidic vacuolar compartment, causing relocation of lysosomal enzymes to the cytosol. Exposure to AcLDL resulted in its uptake with enlargement of the lysosomal apparatus, but the stability of the lysosomal membranes was not changed.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-65822 (URN)10.1161/​01.ATV.18.2.177 (DOI)9484981 (PubMedID)
Available from: 2011-02-21 Created: 2011-02-21 Last updated: 2017-12-11Bibliographically approved
2. OxLDL-induced macrophage cytotoxicity is mediated by lysosomal rupture and modified by intralysosomal redox-active iron
Open this publication in new window or tab >>OxLDL-induced macrophage cytotoxicity is mediated by lysosomal rupture and modified by intralysosomal redox-active iron
1998 (English)In: Free radical research, ISSN 1071-5762, E-ISSN 1029-2470, Vol. 29, no 5, 389-98 p.Article in journal (Refereed) Published
Abstract [en]

Oxidized low density lipoprotein (oxLDL) is believed to play a central role in atherogenesis. LDL is oxidized in the arterial intima by mechanisms that are still only partially understood. OxLDL is then taken up by macrophages through scavenger receptor-mediated endocytosis, which then leads to cellular damage, including apoptosis. The complex mechanisms by which oxLDL induces cell injury are mostly unknown. This study has demonstrated that oxLDL-induced damage of macrophages is associated with iron-mediated intralysosomal oxidative reactions, which cause partial lysosomal rupture and ensuing apoptosis. This series of events can be prevented by pre-exposing cells to the iron-chelator, desferrioxamine (DFO), whereas it is augmented by pretreating the cells with a low molecular weight iron complex. Since both DFO and the iron complex would be taken up by endocytosis, and thus directed to the lysosomal compartment, the results suggest that the normal contents of lysosomal low molecular weight iron may play an important role in oxLDL-induced cell damage, presumably by catalyzing intralysosomal fragmentation of lipid peroxides and the formation of toxic aldehydes and oxygen-centered radicals.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-65821 (URN)10.1080/10715769800300431 (DOI)9925031 (PubMedID)
Available from: 2011-02-21 Created: 2011-02-21 Last updated: 2017-12-11Bibliographically approved
3. Lysosomal destabilization during macrophage damage induced by cholesterol oxidation products
Open this publication in new window or tab >>Lysosomal destabilization during macrophage damage induced by cholesterol oxidation products
Show others...
2000 (English)In: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 28, no 2, 208-218 p.Article in journal (Refereed) Published
Abstract [en]

We have previously shown that oxidized low-density lipoprotein (LDL) induces damage to the macrophage lysosomal membranes, with ensuing leakage of lysosomal contents and macrophage cell death. Cholesterol oxidation products (ChOx) have been reported to be the major cytotoxic components of oxidized LDL/LDL and also to stimulate cholesterol accumulation in vascular cells. In the present study, we characterized the initial events during macrophage damage induced by cholesterol oxidation products (ChOx). Within 24 h of exposure, ChOx caused lysosomal destabilization, release to the cytosol of the lysosomal marker-enzyme cathepsin D, apoptosis, and postapoptotic necrosis. Enhanced autophagocytosis and chromatin margination was found 12 h after the exposure to ChOx, whereas apoptosis and postapoptotic necrosis was pronounced 24 and 48 h after the exposure. Some lysosomal vacuoles were then filled with degraded cellular organelles, indicating phagocytosis of apoptotic bodies by surviving cells. Because caspase-3 activation was detected in the ChOx-exposed cells, lysosomal destabilization may associate with the leakage of lysosomal enzymes, and activation of the caspase cascade. MnSOD mRNA levels were markedly increased after 24 h of exposure to ChOx, suggesting associated induction of mitochondrial protection repair or turnover. We conclude that ChOx-induced damage to lysosomes and mitochondria are sequelae to the cascade of oxysterol cytotoxic events. The early disruption of lysosomes induced by ChOx, with resultant autophagocytosis may be a critical event in apoptosis and/or necrosis of macrophages/foam cells during the development of atherosclerotic lesions.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27688 (URN)10.1016/S0891-5849(99)00220-8 (DOI)12426 (Local ID)12426 (Archive number)12426 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
4. Apoptotic Death of Inflammatory Cells in Human Atheroma
Open this publication in new window or tab >>Apoptotic Death of Inflammatory Cells in Human Atheroma
2001 (English)In: Arteriosclerosis, Thrombosis and Vascular Biology, ISSN 1079-5642, E-ISSN 1524-4636, Vol. 21, no 7, 1124-1130 p.Article in journal (Refereed) Published
Abstract [en]

Although the accumulation of cholesterol and other lipidic material is unquestionably important in atherogenesis, the reasons why this material progressively accumulates, rather than being effectively cleared by phagocytic cells such as macrophages, are not completely understood. We hypothesize that atheromatous lesions may represent "death zones" that contain toxic materials such as oxysterols and in which monocytes/macrophages become dysfunctional and apoptotic. Indeed, cathepsins B and L, normally confined to the lysosomal compartment, are present in the cytoplasm and nuclei of apoptotic (caspase-3-positive) macrophages within human atheroma. The possible involvement of oxysterols is suggested by experiments in which cultured U937 and THP-1 cells exposed to 7-oxysterols similarly undergo marked lysosomal destabilization, caspase-3 activation, and apoptosis. Like macrophages within atheroma, intralysosomal cathepsins B and L are normally present in the cytoplasm and nuclei of these oxysterol-exposed cells. Lysosomal destabilization, cathepsin release, and apoptosis may be causally related, because inhibitors of cathepsins B and L suppress oxysterol-induced apoptosis. Thus, toxic materials such as 7-oxysterols in atheroma may impair the clearance of cholesterol and other lipidic material by fostering the apoptotic death of phagocytic cells, thereby contributing to further development of atherosclerotic lesions.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27724 (URN)10.1161/​hq0701.092145 (DOI)12463 (Local ID)12463 (Archive number)12463 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
5. Induction of cell death by the lysosomotropic detergent MSDH
Open this publication in new window or tab >>Induction of cell death by the lysosomotropic detergent MSDH
Show others...
2000 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 470, no 1, 35-39 p.Article in journal (Refereed) Published
Abstract [en]

Controlled lysosomal rupture was initiated in lysosome-rich, macrophage-like cells by the synthetic lysosomotropic detergent, O-methyl-serine dodecylamide hydrochloride (MSDH). When MSDH was applied at low concentrations, resulting in partial lysosomal rupture, activation of pro-caspase-3-like proteases and apoptosis followed after some hours. Early during apoptosis, but clearly secondary to lysosomal destabilization, the mitochondrial transmembrane potential declined. At high concentrations, MSDH caused extensive lysosomal rupture and necrosis. It is suggested that lysosomal proteases, if released to the cytosol, may cause apoptosis directly by pro-caspase activation and/or indirectly by mitochondrial attack with ensuing discharge of pro-apoptotic factors.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27689 (URN)10.1016/S0014-5793(00)01286-2 (DOI)12427 (Local ID)12427 (Archive number)12427 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved

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