Functional expression of the P-glycoproteinmdr in primary cultures of bovine cerebral capillary endothelial cells

The P-glycoproteinmdr is expressed not only in tumoral cells, but also in nontransformed cells, including the specialized endothelial cells of brain capillaries which build up the blood-brain barrier. Since all previously identified blood-brain barrier markers are rapidly lost when cerebral capillary endothelial cells are maintained in primary culture, we have investigated whether P-glycoprotein (P-gp) would follow the same rule, in order to address the influence of the cerebral environment on the specific P-gp expression in the brain endothelium. As compared to freshly isolated purified cerebral capillaries, P-glycoprotein was detected by immunochemistry at a high level in 5–7 day primary cultures. In our culture conditions, P-glycoprotein was immunodetected at a lower molecular weight than that found in freshly isolated capillaries. Enzymatic deglycosylation led to the same 130 kDa protein for both fresh and cultured samples, suggesting that P-gp post-translational modifications were altered in primary cultures. However, studies on the uptake and efflux of the P-gp substrate [³H]vinblastine, and on the effect of variousmdr reversing agents on the uptake and efflux, clearly indicated that the efflux pump function of the P-glycoprotein was maintained in primary cultures of bovine cerebral capillary endothelial cells. P-Glycoprotein may thus represent the first blood-brain barrier marker which is maintained in cerebral endothelial cells cultured in the absence of factors originating from the brain parenchyma.Abbreviations BBB blood-brain barrier - BCEC brain capillary endothelial cells - -GT -glutamyltranspeptidase - HBSS Hank's balanced salt solution - Mab monoclonal antibody - mdr multidrug resistance - P-gp P-glycoprotein     

Blood-Brain Barrier Effects of the Fusarium Mycotoxins Deoxynivalenol, 3 Acetyldeoxynivalenol,and Moniliformin and Their Transfer to the Brain

Background

Secondary metabolites produced by Fusarium fungi frequently contaminate food and feed and have adverse effects on human and animal health. Fusarium mycotoxins exhibit a wide structural and biosynthetic diversity leading to different toxicokinetics and toxicodynamics. Several studies investigated the toxicity of mycotoxins, focusing on very specific targets, like the brain. However, it still remains unclear how fast mycotoxins reach the brain and if they impair the integrity of the blood-brain barrier. This study investigated and compared the effects of the Fusarium mycotoxins deoxynivalenol, 3-acetyldeoxynivalenol and moniliformin on the blood-brain barrier. Furthermore, the transfer properties to the brain were analyzed, which are required for risk assessment, including potential neurotoxic effects.

Methods

Primary porcine brain capillary endothelial cells were cultivated to study the effects of the examined mycotoxins on the blood-brain barrier in vitro. The barrier integrity was monitored by cellular impedance spectroscopy and ¹⁴C radiolabeled sucrose permeability measurements. The distribution of the applied toxins between blood and brain compartments of the cell monolayer was analyzed by high performance liquid chromatography-mass spectrometry to calculate transfer rates and permeability coefficients.

Results

Deoxynivalenol reduced the barrier integrity and caused cytotoxic effects at 10 μM concentrations. Slight alterations of the barrier integrity were also detected for 3-acetyldeoxynivalenol. The latter was transferred very quickly across the barrier and additionally cleaved to deoxynivalenol. The transfer of deoxynivalenol and moniliformin was slower, but clearly exceeded the permeability of the negative control. None of the compounds was enriched in one of the compartments, indicating that no efflux transport protein is involved in their transport.     

Hydrogen Sulfide Protects HUVECs against Hydrogen Peroxide Induced Mitochondrial Dysfunction and Oxidative Stress

Background

Hydrogen sulfide (H₂S) has been shown to have cytoprotective effects in models of hypertension, ischemia/reperfusion and Alzheimer''s disease. However, little is known about its effects or mechanisms of action in atherosclerosis. Therefore, in the current study we evaluated the pharmacological effects of H₂S on antioxidant defenses and mitochondria protection against hydrogen peroxide (H₂O₂) induced endothelial cells damage.

Methodology and Principal Findings

H₂S, at non-cytotoxic levels, exerts a concentration dependent protective effect in human umbilical vein endothelial cells (HUVECs) exposed to H₂O₂. Analysis of ATP synthesis, mitochondrial membrane potential (ΔΨm) and cytochrome c release from mitochondria indicated that mitochondrial function was preserved by pretreatment with H₂S. In contrast, in H₂O₂ exposed endothelial cells mitochondria appeared swollen or ruptured. In additional experiments, H₂S was also found to preserve the activities and protein expressions levels of the antioxidants enzymes, superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase in H₂O₂ exposed cells. ROS and lipid peroxidation, as assessed by measuring H₂DCFDA, dihydroethidium (DHE), diphenyl-l-pyrenylphosphine (DPPP) and malonaldehyde (MDA) levels, were also inhibited by H₂S treatment. Interestingly, in the current model, D, L-propargylglycine (PAG), a selective inhibitor of cystathionine γ-lyase (CSE), abolished the protective effects of H₂S donors.

Innovation

This study is the first to show that H₂S can inhibit H₂O₂ mediated mitochondrial dysfunction in human endothelial cells by preserving antioxidant defences.

Significance

H₂S may protect against atherosclerosis by preventing H₂O₂ induced injury to endothelial cells. These effects appear to be mediated via the preservation of mitochondrial function and by reducing the deleterious effects of oxidative stress.     

Negative regulation of mitochondrial VDAC channels by C-Raf kinase

Background

Growth of cancer cells results from the disturbance of positive and negative growth control mechanisms and the prolonged survival of these genetically altered cells due to the failure of cellular suicide programs. Genetic and biochemical approaches have identified Raf family serine/threonine kinases B-Raf and C-Raf as major mediators of cell survival. C-Raf cooperates with Bcl-2/Bcl-X_L in suppression of apoptosis by a mechanism that involves targeting of C-Raf to the outer mitochondrial membrane and inactivation of the pro-apoptotic protein Bad. However, apoptosis suppression by C-Raf also occurs in cells lacking expression of Bad or Bcl-2.

Results

Here we show that even in the absence of Bcl-2/Bcl-X_L, mitochondria-targeted C-Raf inhibits cytochrome c release and caspase activation induced by growth factor withdrawal. To clarify the mechanism of Bcl-2 independent survival control by C-Raf at the mitochondria a search for novel mitochondrial targets was undertaken that identified voltage-dependent anion channel (VDAC), a mitochondrial protein (porin) involved in exchange of metabolites for oxidative phosphorylation. C-Raf forms a complex with VDAC in vivo and blocks reconstitution of VDAC channels in planar bilayer membranes in vitro.

Conclusion

We propose that this interaction may be responsible for the Raf-induced inhibition of cytochrome c release from mitochondria in growth factor starved cells. Moreover, C-Raf kinase-induced VDAC inhibition may regulate the metabolic function of mitochondria and mediate the switch to aerobic glycolysis that is common to cancer cells.     

Uptake mechanism of ApoE-modified nanoparticles on brain capillary endothelial cells as a blood-brain barrier model

Background

The blood-brain barrier (BBB) represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE) appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood.

Methodology/Principal Findings

In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor.

Conclusions/Significance

This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier.     

Checkpoint control on mitochondrial division inCyanidioschyzon merolae

Summary It is generally accepted that mitochondria proliferate by division. However, since the apparatus for mitochondrial division was discovered only recently, the basic mechanism of mitochondrial division remains poorly understood. The unicellular red algaCyanidioschyzon merolae is the only organism in which the existence of the apparatus for mitochondrial division (mitochondrion-dividing ring) has been proved by electron microscopy. Since mitochondrial division, mitosis, and cytokinesis regularly occurred in that order, we can assume that tight linkage exists between mitochondrial division and the mitotic cycle. To examine this assumption, we performed experiments with aphidicolin, a specific inhibitor of DNA polymerase , using cells that had been synchronized by a 12 h light/12 h dark treatment. The effects of aphidicolin onC. merolae cells were examined by both epifluorescence and electron microscopy. When cells synchronized at the S phase were treated with aphidicolin, neither mitosis nor cytokinesis occurred. Epifluorescence microscopy after staining with 3,3-dihexyloxacarbocyanine iodide (DiOC₆; a mitochondrion-specific fluorochrome) revealed that mitochondrial division was also completely inhibited. Nevertheless, electron-microscopic examination of the aphidicolin-treated cells clearly revealed the presence of a mitochondrion-dividing ring in mitochondria in all cells examined, in spite of the absence of mitochondrial division. Microbodies, which might be related to mitochondrial division inC. merolae, also failed to divide and became attached to the mitochondrion-dividing rings. These results imply the presence of a checkpoint control mechanism that inhibits division of mitochondria and microbodies in the absence of the synthesis of cell-nuclear DNA.Abbreviation DiOC₆ 3,3-dihexyloxacarbocyanine iodide     

Cytofluorometric quantitation of apoptosis-driven inner mitochondrial membrane permeabilization

The mitochondrial matrix can be specifically labeled by loading cells with calcein and simultaneous quenching of the non-mitochondrial calcein fluorescence with cobalt (Co²⁺). Positive staining of mitochondria thus requires that the inner mitochondrial membrane functions as a barrier separating calcein (within the matrix) from Co²⁺ (outside of the matrix). Upon induction of apoptosis, such calcein/Co²⁺-labeled cells, demonstrate a decrease in the overall calcein fluorescence resulting from inner mitochondrial membrane permeabilization. This decrease can be quantified by cytofluorometry and can be dissociated from other apoptosis-associated mitochondrial perturbations such as the loss of the mitochondrial transmembrane potential ( _m ), the local overproduction of reactive oxygen species, and the mitochondrial release of cytochrome c. In some paradigms of apoptosis the loss of calcein/Co²⁺ (CC) staining can be dissociated from the _m loss, both of which may occur in a caspase-dependent or caspase-independent fashion, depending on the apoptosis inducer. Importantly, inner membrane permeabilization to CC may occur without a permanent _m dissipation in apoptosis, suggesting that transient permeabilization events could participate at the apoptotic cascade. Altogether, our data demonstrate that inner mitochondrial membrane permeabilization constitutes an early event in the apoptotic cascade.     

Denatonium inhibits growth and induces apoptosis of airway epithelial cells through mitochondrial signaling pathways

Background

Denatonium, a widely used bitter agonist, activates bitter taste receptors on many cell types and plays important roles in chemical release, ciliary beating and smooth muscle relaxation through intracellular Ca²⁺-dependent pathways. However, the effects of denatonium on the proliferation of airway epithelial cells and on the integrity of cellular components such as mitochondria have not been studied. In this study, we hypothesize that denatonium might induce airway epithelial cell injury by damaging mitochondria.

Methods

Bright-field microscopy, cell counting kit-8 (CCK-8) assay and flow cytometry analysis were used to examine cellular morphology, proliferation and cell cycle, respectively. Transmission electron microscopy (TEM) was used to examine mitochondrial integrity. JC-1 dye and western blotting techniques were used to measure mitochondrial membrane potential and protein expression, respectively.

Results

For airway epithelial cells, we observed that denatonium significantly effects cellular morphology, decreases cell proliferation and reduces the number of cells in S phase in a dose-dependent manner. TEM analysis demonstrated that denatonium causes large amplitude swelling of mitochondria, which was confirmed by the loss of mitochondrial membrane potential, the down-regulation of Bcl-2 protein and the subsequent enhancement of the mitochondrial release of cytochrome c and Smac/DIABLO after denatonium treatment.

Conclusions

In this study, we demonstrated for the first time that denatonium damages mitochondria and thus induces apoptosis in airway epithelial cells.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0183-9) contains supplementary material, which is available to authorized users.     

Mitofusin 1 is degraded at G<Subscript>2</Subscript>/M phase through ubiquitylation by MARCH5

Background

Mitochondria exhibit a dynamic morphology in cells and their biogenesis and function are integrated with the nuclear cell cycle. In mitotic cells, the filamentous network structure of mitochondria takes on a fragmented form. To date, however, whether mitochondrial fusion activity is regulated in mitosis has yet to be elucidated.

Findings

Here, we report that mitochondria were found to be fragmented in G₂ phase prior to mitotic entry. Mitofusin 1 (Mfn1), a mitochondrial fusion protein, interacted with cyclin B1, and their interactions became stronger in G₂/M phase. In addition, MARCH5, a mitochondrial E3 ubiquitin ligase, reduced Mfn1 levels and the MARCH5-mediated Mfn1 ubiquitylation were enhanced in G₂/M phase.

Conclusions

Mfn1 is degraded through the MARCH5-mediated ubiquitylation in G₂/M phase and the cell cycle-dependent degradation of Mfn1 could be facilitated by interaction with cyclin B1/Cdk1 complexes.

     

Role of endogenous and exogenous antioxidants in the defence against functional damage and lipid peroxidation in rat liver mitochondria

Mitochondria are cellular organelles where the generation of reactive oxygen species may be high. They are, however, effectively protected by their high capacities of antioxidative systems, as enzymes and either water or lipid soluble low molecular weight antioxidants.These antioxidative defence systems can be effectively regenerated after or during an oxidative stress as long as the mitochondria are in an energized state. Energization of mitochondria mainly depends on the availability of suitable respiratory substrates which can provide hydrogen for the reduction of either the glutathione- or -tocopherol-system, since GSH is regenerated by glutathione reductase with the substrate NADPH and the -tocopheroxyl-radical likely by reduced coenzyme Q. It was shown that mitochondria do not undergo damages as long as they can keep a high energy state. The delicate balance between prooxidative/antioxidative activities can be shifted towards oxidation, if experimentally prooxidants were added. After exhaustion of the antioxidative defence systems damages of rnitochondrial functions become expressed followed by membrane injuries along with the oxidation and degradation of mitochondrial lipids and proteins leading finally to the total degradation of the mitoc hondria.Extramitochondrial antioxidants may assist the mitochondrial antioxidative defence systems in a complex way, whereby particularly ascorbic acid can act both as prooxidant and as antioxidant. (Mol Cell Biochem 174: 199–205, 1997)     

Evidence for Mitochondrial Respiratory Deficiency in Rat Rhabdomyosarcoma Cells

Background

Mitochondria can sense signals linked to variations in energy demand to regulate nuclear gene expression. This retrograde signaling pathway is presumed to be involved in the regulation of myoblast proliferation and differentiation. Rhabdomyosarcoma cells are characterized by their failure to both irreversibly exit the cell cycle and complete myogenic differentiation. However, it is currently unknown whether mitochondria are involved in the failure of rhabdomyosarcoma cells to differentiate.

Methodology/Principal Findings

Mitochondrial biogenesis and metabolism were studied in rat L6E9 myoblasts and R1H rhabdomyosacoma cells during the cell cycle and after 36 hours of differentiation. Using a combination of flow cytometry, polarographic and molecular analyses, we evidenced a marked decrease in the cardiolipin content of R1H cells cultured in growth and differentiation media, together with a significant increase in the content of mitochondrial biogenesis factors and mitochondrial respiratory chain proteins. Altogether, these data indicate that the mitochondrial inner membrane composition and the overall process of mitochondrial biogenesis are markedly altered in R1H cells. Importantly, the dysregulation of protein-to-cardiolipin ratio was associated with major deficiencies in both basal and maximal mitochondrial respiration rates. This deficiency in mitochondrial respiration probably contributes to the inability of R1H cells to decrease mitochondrial H₂O₂ level at the onset of differentiation.

Conclusion/Significance

A defect in the regulation of mitochondrial biogenesis and mitochondrial metabolism may thus be an epigenetic mechanism that may contribute to the tumoral behavior of R1H cells. Our data underline the importance of mitochondria in the regulation of myogenic differentiation.     

Biochemical characteristics of primary and passaged cultures of primate brain microvessel endothelial cells

Rhesus macaque monkey brain microvessel endothelial cells (BMECs) were isolated and grown in culture in an effort to establish an appropriate primate in vitro model of the endothelial component of the blood-brain barrier. The presence of Factor VIII antigen, alkaline phosphatase, -glutamyl transpeptidase, lactate dehydrogenanse, total protein, and the passive permeability properties was documented for both primary and passaged cultures. Primate BMECs were shown to exhibit similar morphological and biochemical properties described for other BMEC culture systems derived from other species. In addition, the passaged primate BMECs were particularly notable for the changes in enzyme activities and total protein that parallel age-dependent changes in brain capillary endothelia. This study provides further support for the possible application of BMEC culture systems in investigations of blood-brain barrier functions under normal, aging, and diseased conditions.To whom to address reprint requests. Phone (913)864-3609; FAX (913)864-3578.     

Intracellular targeting of the photoprotein aequorin: A new approach for measuring, in living cells, Ca2+ concentrations in defined cellular compartments

We here present a novel method, based on the targeting of the photoprotein aequorin, for measuring the concentration of Ca²⁺ ions in defined cellular compartments of intact cells. In this contribution we will discuss the application to mitochondria. A chimaeric cDNA was constructed by fusing in frame the aequorin cDNA with that for a mitochondrial protein. The cDNA encoded a mitochondrially-targeted aequorin, composed of a typical mitochondrial targeting signal at the N-terminus and the photoprotein at the C-terminus. The cDNA, inserted in the expression vector pMT2, was co-transfected into bovine endothelial and HeLa cells together with the selectable plasmid pSV2-neo and stable transfectants, selected for high aequorin production, were analyzed. In subcellular fractionations, aequorin was shown to be localized in mitochondria; in intact cells, the first direct measurement of mitochondrial free Ca²⁺, [Ca²⁺]_m, were obtained, which showed that [Ca²⁺]_m is low at rest (<0.5>M), but rapidly increases to the micromolar range upon cell stimulation [1]. These data indicate that mitochondria sense very accurately the cytosolic Ca²⁺ concentration ([Ca²⁺]_i), and after cell stimulation [Ca²⁺]_m rises to values capable of activating the Ca²⁺-sensitive mitochondrial dehydrogenases.     

Direct in vitro action of thyroid hormones on mitochondrial RNA-polymerase

The authors show the direct in vitro action of thyroid hormones on RNA-polymerase activity in rat liver mitochondria. 3,5,3 L-triiodothyronine (L-T₃) and 3,5,3,5 L-tetraiodothyronine (L-T₄) stimulate mitochondrial RNA synthesis without either increasing the permeability of preswollen mitochondria or stimulating the synthesis of the triphosphate ribonucleotides (NTP's). Thyroid hormones do not directly depress mitochondrial RNA hydrolysis. Studies carried out with structural analogues of thyroid hormones indicate the structural specifications of the regulating system of the mitochondrial RNA-polymerase. L-T₃ and L-T₄ are also effective in vitro on mitochondria obtained from animals undergoing different hormonal and dietary treatments, with the exceptions of those fed with a hypoprotein diet. Thus, the authors suggest the possible intervention of a specific mitochondrial receptor for L-T₃ and L-T₄.     

Iron deprivation induces apoptosis via mitochondrial changes related to Bax translocation

In order to elucidate the mechanisms involved in apoptosis induction by iron deprivation, we compared cells sensitive (38C13) and resistant (EL4) to apoptosis induced by iron deprivation. Iron deprivation was achieved by incubation in a defined iron-free medium. We detected the activation of caspase-3 as well as the activation of caspase-9 in sensitive cells but not in resistant cells under iron deprivation. Iron deprivation led to the release of cytochrome c from mitochondria into the cytosol only in sensitive cells but it did not affect the cytosolic localization of Apaf-1 in both sensitive and resistant cells. The mitochondrial membrane potential (_m) was dissipated within 24 h in sensitive cells due to iron deprivation. The antiapoptotic Bcl-2 protein was found to be associated with mitochondria in both sensitive and resistant cells and the association did not change under iron deprivation. On the other hand, under iron deprivation we detected translocation of the proapoptotic Bax protein from the cytosol to mitochondria in sensitive cells but not in resistant cells. Taken together, we suggest that iron deprivation induces apoptosis via mitochondrial changes concerning proapoptotic Bax translocation to mitochondria, collapse of the mitochondrial membrane potential, release of cytochrome c from mitochondria, and activation of caspase-9 and caspase-3.     

A novel rice protein family of OsHIGDs may be involved in early signalling of hypoxia-promoted stem growth in deepwater rice

Key message

OsHIGDs was identified as a novel hypoxia-responsive protein family. Among them, OsHIGD2 is characterized as a mitochondrial protein and is related to hypoxia signalling through interacting with mitochondrial proteins of critical functions in reducing cell damages caused by hypoxia.

Abstract

Recent evidence supports ethylene as a key factor in modulating plant responses to submergence stress. Meanwhile, there has been general consent that ethylene is not the only signal for the submergence-induced stem growth. In this study, we confirmed that hypoxia also promotes stem elongation in deepwater rice even in the absence of ethylene. As components of ethylene-independent hypoxia signalling, five HIGD (hypoxia-induced gene domain) protein genes were identified. Among the genes, OsHIGD2 showed the fastest and strongest induction by hypoxia as well as submergence. Co-expression analysis indicated that OsHIGD2 had a simultaneous expression pattern with fermentation-related genes, such as ADH1 (alcohol dehydrogenase 1) and PDC2 (pyruvate decarboxylase 2). Transient expression of OsHIGD2 in leaf epidermal cells of Nicotiana benthamiana provided evidence that the protein is localized to mitochondria. We further identified OsHIGD2-interacting proteins through the yeast two-hybrid assay using OsHIGD2 as bait. As a result, three mitochondrial proteins were discovered that function in the regulation of redox potential or reduction of protein damages caused by reactive oxygen species. In this report, we propose that OsHIGD2 is a mitochondrial protein which takes part in the early stage of hypoxia signalling by interacting with proteins that are related to oxygen utilization.

     

Ascorbate and low concentrations of FeSO4 induce Ca2+-dependent pore in rat liver mitochondria

Oxidative stress is one of the most frequent causes of tissue and cell injury in various pathologies. The molecular mechanism of mitochondrial damage under conditions of oxidative stress induced in vitro with low concentrations of FeSO₄ and ascorbate (vitamin C) was studied. FeSO₄ (1-4 M) added to rat liver mitochondria that were incubated in the presence of 2.3 mM ascorbate induced (with a certain delay) a decrease in membrane potential and high-amplitude swelling. It also significantly decreased the ability of mitochondria to accumulate exogenous Ca²⁺. All the effects of FeSO₄ + ascorbate were essentially prevented by cyclosporin A, a specific inhibitor of the mitochondrial Ca²⁺-dependent pore (also known as the mitochondrial permeability transition). EGTA restored the membrane potential of mitochondria de-energized with FeSO₄ + ascorbate. We hypothesize that oxidative stress induced in vitro with FeSO₄ and millimolar concentrations of ascorbate damages mitochondria by inducing the cyclosporin A-sensitive Ca²⁺-dependent pore in the inner mitochondrial membrane.     

Mitochondria-targeted antioxidant peptide SS31 attenuates high glucose-induced injury on human retinal endothelial cells

Purpose

To investigate the effect of mitochondria-targeted antioxidant peptide SS31 on prevention of high glucose-induced injury on human retinal endothelial cells (HRECs).

Methods

Cultured P3–P5 HRECs were divided into three groups: 5 mM glucose group, 30 mM glucose group and 30 mM glucose co-treated with 100 nM SS31 group. 24 and 48 h after treatment, Annexin V-FITC/PI staining was used to evaluate the survival of HRECs. Overproduction of ROS was assessed by MitoSOX staining under confocal microscope. Change of mitochondrial potential (ΔΨ_m) of HRECs was measured by flow cytometry after JC-1 fluorescent probe staining. Release of cytochrome c was assessed by confocal microscopy and western blot. Expression of caspase-3 and thioredoxin-2 (Trx-2) were measured by western blot and real-time PCR.

Results

Compared to the high glucose group, co-treatment with 100 nM SS31 significantly protected HRECs from high glucose-induced injury, reduced the production of ROS in mitochondria, stabilized ΔΨ_m, decreased the release of cytochrome c from mitochondria to cytoplasm, decreased the expression of caspase-3 and increased the expression of Trx-2 in high glucose-treated HRECs.

Conclusions

SS31 attenuates the high glucose-induced injuries on HRECs by stabilizing ΔΨ_m, decreasing ROS production, preventing the release of cytochrome c from mitochondria, decreasing the expression of caspase-3 and increasing the expression of Trx-2. Our study suggests that SS31 may be as a potential new treatment for diabetic retinopathy and other oxidative stress-related diseases.     

Increase in mitochondrial mass in human fibroblasts under oxidative stress and during replicative cell senescence

Abnormal proliferation of mitochondria generally occurs in muscle of aged individuals and patients with mitochondrial myopathy. An increase in the mitochondrial DNA (mtDNA) copy number has also been observed in aging human tissues. However, the molecular mechanism underlying the increase in mitochondrial mass and mtDNA is still unclear. In a previous study, we demonstrated that sublethal levels of oxidative stress caused an increase in mitochondrial mass in human lung cells. In this communication, we report our recent findings that the mitochondrial mass in human lung fibroblasts (MRC-5) in a later proliferation stage is significantly increased compared to that in the early stages of proliferation. The extent of the increase in mitochondrial mass in the senescent cells was similar to that in cells in the early stages of proliferation that had been treated with low concentrations ( 180 µM) of hydrogen peroxide (H₂O₂). Moreover, we found that the rate of reactive oxygen species (ROS) production was higher in cells in the later proliferation stage compared to cells in the early proliferation stages. A similar phenomenon was also observed in cells in the early proliferation stages under low levels of oxidative stress. On the other hand, the mRNA levels of many nuclear DNA-encoded proteins involved in mitochondrial biogenesis, particularly nuclear respiratory factor-1, were found to increase in cells in later proliferation stages and in cells in early proliferation stages that had been treated with 180 µM H₂O₂. Interestingly, the increase in mitochondrial mass in the cells under oxidative stress could be repressed by treatment with cycloheximide orm-chlorocarbonyl cyanide phenylhydrazone but not by chloramphenicol. Furthermore, the mitochondrial mass of mtDNA-less ° cells was also significantly increased by exposure to low concentrations (e.g. 180 µM) of H₂O₂. These results suggest that the increase in mitochondrial mass in replicative senescent cells may result from an increase in ROS production, and that it is dependent on both de novo synthesis of nuclear DNA-encoded proteins and their import into mitochondria, dictated by the membrane potential of mitochondria.