Impairment of mitochondrial respiratory chain activity in aortic endothelial cells induced by glycated low-density lipoprotein

Coronary artery disease (CAD) is the leading cause of mortality in diabetic patients. Mitochondrial dysfunction and increased production of reactive oxygen species (ROS) are associated with diabetes and CAD. Elevated levels of glycated LDL (glyLDL) were detected in patients with diabetes. Our previous studies demonstrated that glyLDL increased the generation of ROS and altered the activities of antioxidant enzymes in vascular endothelial cells (EC). This study examined the effects of glyLDL on oxygen consumption in mitochondria and the activities of key enzymes in the mitochondrial electron transport chain (ETC) in cultured porcine aortic EC. The results demonstrated that glyLDL treatment significantly impaired oxygen consumption in Complexes I, II/III, and IV of the mitochondrial ETC in EC compared to LDL or vehicle control detected using oxygraphy. Incubation with glyLDL significantly reduced the mitochondrial membrane potential, the NAD⁺/NADH ratio, and the activities of mitochondrial ETC enzymes (NADH-ubiquinone dehydrogenase, succinate cytochrome c reductase, ubiquinone cytochrome c reductase, and cytochrome c oxidase) in EC compared to LDL or control. The abundance of mitochondria-associated ROS and the release of ROS from EC were significantly increased after glyLDL treatment. The findings suggest that glyLDL attenuates the activities of key enzymes in the mitochondrial ETC, decreases mitochondrial oxygen consumption, reduces mitochondrial membrane potential, and increases ROS generation in EC, which potentially contribute to mitochondrial dysfunction in diabetic patients.     

Protective effects of honokiol against oxidized LDL-induced cytotoxicity and adhesion molecule expression in endothelial cells

Honokiol, a compound extracted from Chinese medicinal herb Magnolia officinalis, has several biological effects. However, its protective effects against endothelial injury remain unclarified. In this study, we examined whether honokiol prevented oxidized low-density lipoprotein (oxLDL)-induced vascular endothelial dysfunction. Incubation of oxLDL with honokiol (2.5-20 microM) inhibited copper-induced oxidative modification as demonstrated by diene formation, thiobarbituric acid reactive substances (TBARS) assay and electrophoretic mobility assay. Expression of adhesion molecules (ICAM, VCAM and E-selectin) and endothelial NO synthase (eNOS) affected by oxLDL was investigated by flow cytometry and Western blot. We also measured the production of reactive oxygen species (ROS) using the fluorescent probe 2',7'-dichlorofluorescein acetoxymethyl ester (DCF-AM). Furthermore, several apoptotic phenomena including increased cytosolic calcium, alteration of mitochondrial membrane potential, cytochrome c release and activation of caspase-3 were also investigated. Apoptotic cell death was characterized by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) stain. The results showed that honokiol prevented the copper-induced oxidative modification of LDL. Honokiol also ameliorated the oxLDL-diminished eNOS protein expression and reduced the oxLDL-induced adhesion molecules and the adherence of THP-1 cells to HUVECs. Furthermore, honokiol attenuated the oxLDL-induced cytotoxicity, apoptotic features, ROS generation, intracellular calcium accumulation and the subsequent mitochondrial membrane potential collapse, cytochrome c release and activation of caspase-3. Our results suggest that honokiol may have clinical implications in the prevention of atherosclerotic vascular disease.     

Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis

During apoptosis, the permeabilization of the mitochondrial outer membrane allows the release of cytochrome c, which induces caspase activation to orchestrate the death of the cell. Mitochondria rapidly lose their transmembrane potential (Delta Psi m) and generate reactive oxygen species (ROS), both of which are likely to contribute to the dismantling of the cell. Here we show that both the rapid loss of Delta Psi m and the generation of ROS are due to the effects of activated caspases on mitochondrial electron transport complexes I and II. Caspase-3 disrupts oxygen consumption induced by complex I and II substrates but not that induced by electron transfer to complex IV. Similarly, Delta Psi m generated in the presence of complex I or II substrates is disrupted by caspase-3, and ROS are produced. Complex III activity measured by cytochrome c reduction remains intact after caspase-3 treatment. In apoptotic cells, electron transport and oxygen consumption that depends on complex I or II was disrupted in a caspase-dependent manner. Our results indicate that after cytochrome c release the activation of caspases feeds back on the permeabilized mitochondria to damage mitochondrial function (loss of Delta Psi m) and generate ROS through effects of caspases on complex I and II in the electron transport chain.     

Modified low density lipoproteins decrease the activity and expression of lysosomal acid lipase in human endothelial and smooth muscle cells

Lysosomal acid lipase (LAL), the only lysosomal enzyme involved in the hydrolysis of LDL-cholesteryl esters, is a key regulator of cellular cholesterol and fatty acid homeostasis and its deficiency contributes to the pathophysiology of various diseases. In this study, we questioned whether oxidized or glycated LDL, a common occurrence in atherosclerosis and diabetes, affect the activity and expression of LAL in vascular endothelial cells (EC) and smooth muscle cells (SMC). LAL activity and expression were assayed in cultured human EC and SMC exposed to oxidized LDL (oxLDL), (±)9-hydroxyoctadecadienoic acid-cholesteryl ester (HODE), glycated LDL (gLDL), or native LDL (nLDL) as control, in the presence or absence of LXR or PPAR-gamma agonists. We found that LAL activity and expression were significantly down regulated by oxLDL and HODE in EC, and by gLDL in SMC. The LXR agonist T0901317 reversed the decreased LAL expression in modified LDL- or HODE-exposed EC (P < 0.001) and in gLDL-exposed SMC, whereas PPAR-gamma agonist rosiglitazone induced a low effect only in EC. In conclusion, modified LDL down regulates LAL expression in human EC and SMC by a process involving the LXR signaling pathway. This is the first demonstration that modified LDL modulate LAL expression, in a cell specific manner.     

The influence of dietary lipid composition on liver mitochondria from mice following 1 month of calorie restriction

To investigate the role mitochondrial membrane lipids play in the actions of CR (calorie restriction), C57BL/6 mice were assigned to four groups (control and three 40% CR groups) and the CR groups were fed diets containing soya bean oil (also in the control diet), fish oil or lard. The fatty acid composition of the major mitochondrial phospholipid classes, proton leak and H₂O₂ production were measured in liver mitochondria following 1 month of CR. The results indicate that mitochondrial phospholipid fatty acids reflect the PUFA (polyunsaturated fatty acid) profile of the dietary lipid sources. CR significantly decreased the capacity of ROS (reactive oxygen species) production by Complex III but did not markedly alter proton leak and ETC (electron transport chain) enzyme activities. Within the CR regimens, the CR-fish group had decreased ROS production by both Complexes I and III, and increased proton leak when compared with the other CR groups. The CR-lard group showed the lowest proton leak compared with the other CR groups. The ETC enzyme activity measurements in the CR regimens showed that Complex I activity was decreased in both the CR-fish and CR-lard groups. Moreover, the CR-fish group also had lower Complex II activity compared with the other CR groups. These results indicate that dietary lipid composition does influence liver mitochondrial phospholipid composition, ROS production, proton leak and ETC enzyme activities in CR animals.     

Resveratrol attenuates oxLDL-stimulated NADPH oxidase activity and protects endothelial cells from oxidative functional damages.

trans-Resveratrol (RSV) has been shown to have cardioprotective effect during ischemia-reperfusion through reactive oxygen species (ROS)-scavenging activity. Elevated ROS has been implicated in the initiation and progression of atherosclerosis. The nicotinamide adenine dinucleotide phosphate oxidase (NOX) is a major source of vascular ROS formation. In the present study, we show that exposure of vascular endothelial cells (EC) to oxidized low-density lipoproteins (oxLDL) results in elevations of NOX activity and cellular ROS levels. The oxLDL effects are effectively suppressed by RSV or astringinin (AST), either before or after oxLDL exposure. In this study, we show that RSV or AST treatment appears to suppress NOX activity by reducing the membrane association of gp91(phox) and Rac1, two protein species required for the assembly of active NOX complex. Exposure to RSV or AST protects EC from oxidative functional damages, including antiplatelet activity and mononucleocyte adhesion. In addition, ANG II-induced NOX activation is also attenuated. These results suggest that RSV or AST protects EC from oxLDL-induced oxidative stress by both direct ROS scavenging and inhibition of NOX activity.     

17Beta-estradiol inhibits oxidized low density lipoprotein-induced generation of reactive oxygen species in endothelial cells.

Increase of intracellular reactive oxygen species (ROS) has been proposed to cause endothelial injury, and oxidized LDL (oxLDL) actions are associated with an early increase of ROS. Estrogen protects vascular cells partly via its antioxidant effects and by preventing lipid peroxidation. However, whether it can inhibit oxLDL-induced stimulation of ROS generation in endothelial cells is unknown. We utilized the fluorescent dye (DCFH-DA) to measure ROS generation and compared the stimulant effect of tert-butylhydroperoxide (TBH) and oxLDL in human umbilical vein endothelial cells (HUVECs). We found that TBH, H2O2, and oxLDL rapidly stimulated ROS generation, and in a dose-dependent manner with TBH. A concentration of estrogen effective in preventing lipid peroxidation was employed either by pretreatment of cells 18 h prior to or by direct co-incubation (30 min) with HUVEC and oxLDL. Estrogen (54 microM) pretreatment significantly suppressed both TBH- and oxLDL- induced stimulation of ROS generation. Both 1 and 54 microM concentration of estrogen could directly inhibit oxLDL-induced ROS production in HUVECs. Thus, either 18 h pretreatment or 30 min co-incubation with estrogen reduced stimulated ROS generation, suggesting that both cellular and direct actions of estrogen may be involved.     

Does strong hypertrophic condition induce fast mitochondrial DNA mutation of rabbit heart?

Homo- and heteroplasmic mitochondrial DNA (mtDNA) mutations were observed and identified in an isoproterenol-induced rabbit model of cardiac hypertrophy. Genes encoding proteins essential for catalyzing mitochondrial electron transfer and for generating the proton motive force, such as NADH dehydrogenases (ND2, ND3, ND4, and ND6), cytochrome b, and ATPase 8, showed increased susceptibility for mutation. Specifically, five mutations caused amino acid changes and were located in Complex I and Complex V gene clusters. To our knowledge, this is the first demonstration of a relationship between cardiac hypertrophy induced by a strong sympathetic load and rapid mtDNA mutations.     

Mitochondria,oxidative stress and cell death

In addition to the well-established role of the mitochondria in energy metabolism, regulation of cell death has recently emerged as a second major function of these organelles. This, in turn, seems to be intimately linked to their role as the major intracellular source of reactive oxygen species (ROS), which are mainly generated at Complex I and III of the respiratory chain. Excessive ROS production can lead to oxidation of macromolecules and has been implicated in mtDNA mutations, ageing, and cell death. Mitochondria-generated ROS play an important role in the release of cytochrome c and other pro-apoptotic proteins, which can trigger caspase activation and apoptosis. Cytochrome c release occurs by a two-step process that is initiated by the dissociation of the hemoprotein from its binding to cardiolipin, which anchors it to the inner mitochondrial membrane. Oxidation of cardiolipin reduces cytochrome c binding and results in an increased level of “free” cytochrome c in the intermembrane space. Conversely, mitochondrial antioxidant enzymes protect from apoptosis. Hence, there is accumulating evidence supporting a direct link between mitochondria, oxidative stress and cell death.     

Neuronal Mitochondrial Toxicity of Malondialdehyde: Inhibitory Effects on Respiratory Function and Enzyme Activities in Rat Brain Mitochondria

Malondialdehyde (MDA) is a product of oxidative damage to lipids, amino acids and DNA, and accumulates with aging and diseases. MDA can possibly react with amines so as to modify proteins and inactivate enzymes; it can also modify nucleosides so as to cause mutagenicity. Brain mitochondrial dysfunction is a major contributor to aging and neurodegenerative diseases. We hypothesize that MDA accumulated during aging targets mitochondrial enzymes so as to cause further mitochondrial dysfunction and additional contributions to aging and neurodegeneration. Herein, we investigated the neuronal mitochondrial toxic effects of MDA on mitochondrial respiration and activities of enzymes (mitochondrial complexes I–V, α-ketoglutarate dehydrogenase (KGDH) and pyruvate dehydrogenase (PDH)), in isolated rat brain mitochondria. MDA depressed mitochondrial membrane potential, and also showed a dose-dependent inhibition of mitochondrial complex I- and complex II-linked respiration. Complex I and II, and PDH activities were depressed by MDA at ≥0.2 μmol/mg; KGDH and complex V were inhibited by ≥0.4 and ≥1.6 μmol MDA/mg, respectively. However, MDA did not have any toxic effects on complex III and IV activities over the range 0–2 μmol/mg. MDA significantly elevated mitochondrial reactive oxygen species (ROS) and protein carbonyls at 0.2 and 0.002 μmol/mg, respectively. As for the antioxidant defense system, a high dose of MDA slightly decreased mitochondrial GSH and superoxide dismutase. These results demonstrate that MDA causes neuronal mitochondrial dysfunction by directly promoting generation of ROS and modifying mitochondrial proteins. The results suggest that MDA-induced neuronal mitochondrial toxicity may be an important contributing factor to brain aging and neurodegenerative diseases. Special issue article in honor of Dr. Akitane Mori.     

The protective effects of HDL and its constitutents against neutrophil respiratory burst activation by hypochlorite-oxidized LDL

Hypochlorite-oxidized low-density lipoprotein (oxLDL) possesses a substantial proinflammatory potential by modulating respiratory burst activities of polymorphonuclear neutrophils (PMN). As evaluated by luminol-amplified chemiluminescence (CL) incubation of 10(6) PMN/ml with 70 nM oxLDL was followed by substantial induction of neutrophil oxidant (ROS) generation. We evaluated the inhibitory capacity of high-density lipoprotein (HDL) and its lipid and protein constituents against the activating effects of oxLDL. At a HDL or apolipoprotein AI/LDL protein ratio of 1.0, native HDL decreased the respiratory burst activation by 64%, followed by trypsinized HDL (57%) and native apoAI (43%). The inhibitory effects of native HDL did not require prior incubation with PMN or with oxLDL suggesting an instantaneously acting protective mechanism in the minute range. OxLDL modulated ROS production not only of resting PMN but also that of activated PMN, as indicated by a 14-fold increase in FMLP-stimulated CL response and a 50% decrease in zymosan-mediated CL answer. HDL itself did not protect PMN from activation by FMLP and zymosan. However, it clearly reduced effects of oxLDL on FMLP-activation and slightly counteracted the oxLDL-mediated decrease in zymosan-induced ROS generation. Taken together, these findings may offer new insight into atheroprotective mechanisms of HDL.     

The Contribution of Mitochondrial Respiratory Complexes to the Production of Reactive Oxygen Species

This work was focused on distinguishing the contribution of mitochondrial redox complexesto the production of reactive oxygen species (ROS) during cellular respiration. We were ableto accurately measure, for the first time, the basal production of ROS under uncoupled conditionsby using a very sensitive method, based on the fluorescent probe dichlorodihydrofluoresceindiacetate. The method also enabled the detection of the ROS generated by the oxidation ofthe endogenous substrates in the mitochondrial preparations and could be applied to bothmitochondria and live cells. Contrary to the commonly accepted view that complex III(ubiquinol:cytochrome c reductase) is the major contributor to mitochondrial ROS production, wefound that complex I (NADH-ubiquinone reductase) and complex II (succinate-ubiquinonereductase) are the predominant generators of ROS during prolonged respiration under uncoupledconditions. Complex II, in particular, appears to contribute to the basal production of ROSin cells.     

Membrane topology of beef-heart ubiquinone-cytochrome c reductase (complex III)

The membrane topology of ubiquinone-cytochrome c reductase (EC 1.10.2.2.) has been investigated with photoreactive lipid analogs (Bisson, R., and Montecucco, C. (1981) Biochem. J. 193, 757-763), both in its isolated form and when part of succinate-cytochrome c reductase (Complex II + III). These probes react specifically with those polypeptide chains exposed to lipids, thereby labeling them radioactively. Highly resolving gel electrophoretic conditions have been used to determine the patterns of labeling. Core protein I, cytochrome b, cytochrome c1, and polypeptides VI, VII, VIII, and IX contribute to the lipid-protein boundary of Complex III. Evidence that the interaction between Complex II and Complex III involves their hydrophobic domains is also presented.     

Structural organization of the mitochondrial respiratory chain

Two models exist of the mitochondrial respiratory chain: the model of a random organization of the individual respiratory enzyme complexes and that of a super-complex assembly formed by stable association between the individual complexes. Recently Sch?gger, using digitonin solubilization and Blue Native PAGE produced new evidence of preferential associations, in particular a Complex I monomer with a Complex III dimer, and suggested a model of the respiratory chain (the respirasome) based on direct electron channelling between complexes. Discrimination between the two models is amenable to kinetic testing using flux control analysis. Experimental evidence obtained in beef heart SMP, according to the extension of the Metabolic Control Theory for pathways with metabolic channelling, showed that enzyme associations involving Complex I and Complex III take place in the respiratory chain while Complex IV seems to be randomly distributed, with cytochrome c behaving as a mobile component. Flux control analysis at anyone of the respiratory complexes involved in aerobic succinate oxidation indicated that Complex II and III are not functionally associated in a stable supercomplex. A critical appraisal of the solid-state model of the mitochondrial respiratory chain requires its reconciliation with previous biophysical and kinetic evidence that CoQ behaves as a homogeneous diffusible pool between all reducing enzyme and all oxidizing enzymes: the hypothesis can be advanced that both models (CoQ pool and supercomplexes) are true, by postulating that supercomplexes physiologically exist in equilibrium with isolated complexes depending on metabolic conditions of the cell.     

Mitochondrial regulation of hypoxia-induced increase of adrenomedullin mRNA in HL-1 cells

Hypoxia upregulates the expression of the cardioprotective peptide adrenomedullin in cardiomyocytes. We characterized this pathway in murine HL-1 cardiomyocytes. Inhibition of mitochondrial complexes I, III, and IV largely, but not completely, reduced hypoxic adrenomedullin mRNA increase in gas-impermeable culture plates. Complex III inhibition was also effective in permeable culture plates, so that this effect is unlikely due to intracellular oxygen redistribution, whereas complex I blockade was ineffective in permeable plates. Complex II does not participate in this effect, as shown by chemical and siRNA inactivation. ROS scavenging by nitroblue tetrazolium and general flavoprotein inhibition by diphenyleniodonium nearly abrogated the hypoxic adrenomedullin mRNA increase. Thus, ROS production by flavoproteins is crucial for hypoxic upregulation of adrenomedullin mRNA in murine HL-1 cardiomyocytes. These ROS originate both from the mitochondrial complex III and from additional, presumably extramitochondrial, sources. Mitochondrial oxygen consumption appears to have impact on oxygen availability at these extramitochondrial sensors.     

Generator-specific targets of mitochondrial reactive oxygen species

To understand the role of reactive oxygen species (ROS) in oxidative stress and redox signaling it is necessary to link their site of generation to the oxidative modification of specific targets. Here we have studied the selective modification of protein thiols by mitochondrial ROS that have been implicated as deleterious agents in a number of degenerative diseases and in the process of biological aging, but also as important players in cellular signal transduction. We hypothesized that this bipartite role might be based on different generator sites for “signaling” and “damaging” ROS and a directed release into different mitochondrial compartments. Because two main mitochondrial ROS generators, complex I (NADH:ubiquinone oxidoreductase) and complex III (ubiquinol:cytochrome c oxidoreductase; cytochrome bc₁ complex), are known to predominantly release superoxide and the derived hydrogen peroxide (H₂O₂) into the mitochondrial matrix and the intermembrane space, respectively, we investigated whether these ROS generators selectively oxidize specific protein thiols. We used redox fluorescence difference gel electrophoresis analysis to identify redox-sensitive targets in the mitochondrial proteome of intact rat heart mitochondria. We observed that the modified target proteins were distinctly different when complex I or complex III was employed as the source of ROS. These proteins are potential targets involved in mitochondrial redox signaling and may serve as biomarkers to study the generator-dependent dual role of mitochondrial ROS in redox signaling and oxidative stress.     

An example of Leber hereditary optic neuropathy not involving a mutation in the mitochondrial ND4 gene.

A large Australian family afflicted with Leber's Hereditary Optic Neuropathy (LHON) is analyzed at the nucleotide sequence level in this report. Biochemical assays of platelet mitochondria isolated from members of this family have demonstrated a significant decrease in the specific activity of Complex I (NADH-ubiquinol oxidoreductase) of the electron transport chain. It is shown here, however, that neither this biochemical lesion nor the optic neuropathy are due to the mutation at nucleotide position 11,778 of the mitochondrial ND4 gene first identified by Wallace et al. in several LHON pedigrees. Furthermore, extensive DNA sequencing studies reveal no candidate mutations within the mitochondrial ND3 gene, the ND4L/ND4 genes, or the contiguous tRNA genes. These studies provide the first direct evidence that not all LHON lineages--even those associated with a biochemical defect in mitochondrial respiratory chain Complex I--carry a mutation in the ND4 gene. Members of the Australian LHON family exhibit neurological abnormalities in addition to the well-characterized ophthalmological changes. It is hypothesized that LHON may be a syndrome or set of related diseases in which the clinical abnormalities are a function, at least in part, of the mitochondrial Complex I gene in which the proximate mutation occurs.     

Impaired OXPHOS complex III in breast cancer

We measured the mitochondrial oxidative phosphorylation (mtOXPHOS) activities of all five complexes and determined the activity and gene expression in detail of the Complex III subunits in human breast cancer cell lines and primary tumors. Our analysis revealed dramatic differences in activity of complex III between normal and aggressive metastatic breast cancer cell lines. Determination of Complex III subunit gene expression identified over expression and co-regulation of UQCRFS1 (encoding RISP protein) and UQCRH (encoding Hinge protein) in 6 out of 9 human breast tumors. Analyses of UQCRFS1/RISP expression in additional matched normal and breast tumors demonstrated an over expression in 14 out of 40 (35%) breast tumors. UQCRFS1/RISP knockdown in breast tumor cell line led to decreased mitochondrial membrane potential as well as a decrease in matrigel invasion. Furthermore, reduced matrigel invasion was mediated by reduced ROS levels coinciding with decreased expression of NADPH oxidase 2, 3, 4 and 5 involved in ROS production. These studies provide direct evidence for contribution of impaired mtOXPHOS Complex III to breast tumorigenesis.     

Mitochondrial myopathy involving ubiquinol-cytochrome c oxidoreductase (complex III) identified by immunoelectron microscopy

The distribution of respiratory chain complexes in bovine heart and human muscle mitochondria has been explored by immunoelectron microscopy with antibodies made against bovine heart mitochondrial proteins in conjunction with protein A-colloidal gold (12-nm particles). The antibodies used were made against NADH-coenzyme Q reductase (complex I), ubiquinol cytochrome c oxidoreductase (complex III), cytochrome c oxidase, core proteins isolated from complex III and the non-heme iron protein of complex III. Labeling of bovine heart tissue with any of these antibodies gave gold particles randomly distributed along the mitochondrial inner membrane. The labeling of muscle tissue from a patient with a mitochondrial myopathy localized by biochemical analysis to complex III was quantitated and compared with the labeling of human control muscle tissue. Complex I and cytochrome c oxidase antibodies reacted to the same level in myopathic and normal muscle samples. Antibodies to complex III or its components reacted very poorly to the patient's tissue but strongly to control muscle samples. Immunoelectron microscopy using respiratory chain antibodies appears to be a promising approach to the diagnosis and characterization of mitochondrial myopathies when only limited amounts of tissue are available for study.