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N S Chandel

Publications and source records attributed to N S Chandel.

At least 19 recordsLinked to original sources
PubMed2004

The alpha4 laminin subunit regulates endothelial cell survival.

The alpha4 laminin subunit is a major structural component of assembling basement membranes of endothelial cells. We have been investigating its functions with regard to endothelial cell survival. An anti-laminin alpha4 antibody (2A3), directed against the G domain of the alpha4 laminin subunit of laminins-8 and -9, inhibits proliferation and enhances apoptosis of endothelial cells when cells are maintained in vitro. Activation of caspases-9 and -3 plays a role in 2A3 antibody-induced apoptosis, since inhibitors specific for these caspases and overexpression of the anti-apoptotic protein Bcl-X(L), but not c-FLIP, inhibit 2A3 antibody-triggered endothelial cell death. Extracellular matrix is known to play a role in regulating programmed cell death in an integrin-dependent fashion. The alpha4 laminin subunit conforms to this idea since activation of beta1 integrin subunits on endothelial cells blocks the ability of 2A3 antibody to induce endothelial cell death. In summary, our data indicate that complexes composed of alpha4 laminin/beta1 subunit-containing integrins at the cell surface support endothelial cell survival. Furthermore, we propose that antagonists of alpha4 laminin function, including antibody 2A3, have value as angiogenesis inhibitors in a clinical setting where blocking aberrant growth of blood vessel by triggering apoptosis of endothelial cells may be therapeutic.

Antibodies↗
PubMed2002

Oxygen deprivation induced cell death: an update.

Mammalian cells have multiple responses to low or zero oxygen concentrations. In the complete absence of oxygen, cells undergo cell death through apoptosis, and not necrosis. Apoptotic signaling during oxygen deprivation occurs through the release of cytochrome c and apaf-1 mediated caspase-9 activation. The upstream regulators of cytochrome c release are the Bcl-2 family members. Pro-apoptotic Bcl-2 family members such as bax or bak are clearly required to initiate cytochrome c/apaf-1/caspase-9 mediated cell death during oxygen deprivation. Here we review what is currently known oxygen deprivation induced cell death and speculate about initiating mechanisms resulting in the activation of pro-apoptotic Bcl-2 family members.

Animals↗
PubMed2001

Reactive oxygen species are downstream products of TRAF-mediated signal transduction.

Members of the TNFR (tumor necrosis factor receptor) superfamily are involved in regulating activation and differentiation of cells as well as cell survival and programmed cell death/apoptosis. Multimerization of TNFRs can lead to recruitment of TRAFs (TNFR-associated factors) by the receptors resulting in activation of kinases and transcription factors, such as c-Jun N-terminal kinase and nuclear factor kappaB (NF-kappaB). Signal transduction triggered by TNF-alpha also induces an increase in intracellular reactive oxygen species (ROS). ROS have been suggested to play a role in NF-kappaB activation, which is thought to promote cell survival. However, oxidation of proteins and lipids by ROS can also result in apoptosis. The processes generating intracellular ROS and the mechanism(s) regulating the cellular redox status have not been fully elucidated. We investigated whether TRAFs play a role in controlling intracellular ROS levels. Our results indicate that recruitment of TRAFs to the plasma membrane of human embryonic kidney (HEK) 293 cells is crucial for activation of signaling pathways, which regulate ROS production in mitochondria. TRAF-mediated changes in ROS levels enhanced NF-kappaB activation but were not dependent on NF-kappaB-inducing kinase. Consistent with its anti-apoptotic function, Bcl-x(L) interfered with TRAF-mediated ROS generation but not NF-kappaB activation. Taken together, our results suggest a novel role of TRAFs in signal transduction pathways triggered by TNFR-related proteins, which balance cell survival and apoptosis by regulating the electron transport in mitochondria.

Apoptosis↗
PubMed2001

Model for hypoxic pulmonary vasoconstriction involving mitochondrial oxygen sensing.

We tested whether mitochondria function as the O(2) sensor underlying hypoxic pulmonary vasoconstriction (HPV). In buffer-perfused rat lungs, rotenone, myxothiazol, and diphenyleneiodonium, which inhibit mitochondria in the proximal region of the electron transport chain (ETC), abolished HPV without attenuating the response to U46619. Cyanide and antimycin A inhibit electron transfer in the distal region of the ETC, but they did not abolish HPV. Cultured pulmonary artery (PA) myocytes contract in response to hypoxia or to U46619. The hypoxic response was abolished while the response to U46619 was maintained in mutant (rho(0)) PA myocytes lacking a mitochondrial ETC. To test whether reactive oxygen species (ROS) derived from mitochondria act as signaling agents in HPV, the antioxidants pyrrolidinedithiocarbamate and ebselen and the Cu,Zn superoxide dismutase inhibitor diethyldithiocarbamate were used. These abolished HPV without affecting contraction to U46619, suggesting that ROS act as second messengers. In cultured PA myocytes, oxidation of intracellular 2',7'-dichlorofluorescin diacetate (DCFH) dye increased under 2% O(2), indicating that myocytes increase their generation of H(2)O(2) during hypoxia. This was attenuated by myxothiazol, implicating mitochondria as the source of increased ROS during HPV. These results indicate that mitochondrial ATP is not required for HPV, that mitochondria function as O(2) sensors during hypoxia, and that ROS generated in the proximal region of the ETC act as second messengers in the response.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2000

Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1alpha during hypoxia: a mechanism of O2 sensing.

During hypoxia, hypoxia-inducible factor-1alpha (HIF-1alpha) is required for induction of a variety of genes including erythropoietin and vascular endothelial growth factor. Hypoxia increases mitochondrial reactive oxygen species (ROS) generation at Complex III, which causes accumulation of HIF-1alpha protein responsible for initiating expression of a luciferase reporter construct under the control of a hypoxic response element. This response is lost in cells depleted of mitochondrial DNA (rho(0) cells). Overexpression of catalase abolishes hypoxic response element-luciferase expression during hypoxia. Exogenous H(2)O(2) stabilizes HIF-1alpha protein during normoxia and activates luciferase expression in wild-type and rho(0) cells. Isolated mitochondria increase ROS generation during hypoxia, as does the bacterium Paracoccus denitrificans. These findings reveal that mitochondria-derived ROS are both required and sufficient to initiate HIF-1alpha stabilization during hypoxia.

Androstadienes↗
PubMed2000

Redox regulation of p53 during hypoxia.

The transcription factor p53 can induce growth arrest or death in cells. Tumor cells that develop mutations in p53 demonstrate a diminished apoptotic potential, which may contribute to growth and tumor metastasis. Cellular levels of p53 are stabilized during hypoxia. The present study tested the hypothesis that reactive oxygen species (ROS) released from mitochondria regulate the cytosolic redox state and are required for the stabilization of p53 protein levels in response to hypoxia. Our results indicate that hypoxia (1.5% O2) increases mitochondrial ROS generation and increases p53 protein levels in human breast carcinoma MCF-7 cells and in normal human diploid fibroblast IMR-90 cells. MCF-7 cells depleted of their mitochondrial DNA (rho(o) cells) failed to stabilize p53 protein levels during hypoxia. The antioxidant N-acetylcysteine and the Cu/Zn superoxide dismutase inhibitor diethyldithiocarbamic acid abolished the hypoxia-induced increases in ROS and p53 levels. Rotenone, an inhibitor of mitochondrial complex I, and 4,4'-diisothiocyanato-stilbene-2,2'-disulfonate, a mitochondrial anion channel inhibitor, also abolished the increase in ROS signal and p53 levels during hypoxia. The p53-dependent gene p21WAF1/CIP1 was also induced by hypoxia in both MCF-7 and IMR-90 cells without affecting the growth rate of either cell line. In contrast, both cell lines exhibited increases in p21WAF1/CIP1 expression and growth arrest after gamma irradiation. Primary chick cardiac myocytes and murine embryonic fibroblasts also showed an increase in p53 protein levels in response to hypoxia without cell death or growth arrest. These results indicate that mitochondria regulate p53 protein levels during hypoxia through a redox-dependent mechanism involving ROS. Despite p53-induction, hypoxia alone does not cause either growth arrest or cell death.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2000

Role of oxidants in NF-kappa B activation and TNF-alpha gene transcription induced by hypoxia and endotoxin.

The transcription factor NF-kappa B stimulates the transcription of proinflammatory cytokines including TNF-alpha. LPS (endotoxin) and hypoxia both induce NF-kappa B activation and TNF-alpha gene transcription. Furthermore, hypoxia augments LPS induction of TNF-alpha mRNA. Previous reports have indicated that antioxidants abolish NF-kappa B activation in response to LPS or hypoxia, which suggests that reactive oxygen species (ROS) are involved in NF-kappa B activation. This study tested whether mitochondrial ROS are required for both NF-kappaB activation and the increase in TNF-alpha mRNA levels during hypoxia and LPS. Our results indicate that hypoxia (1.5% O2) stimulates NF-kappa B and TNF-alpha gene transcription and increases ROS generation as measured by the oxidant sensitive dye 2',7'-dichlorofluorescein diacetate in murine macrophage J774.1 cells. The antioxidants N-acetylcysteine and pyrrolidinedithiocarbamic acid abolished the hypoxic activation of NF-kappa B, TNF-alpha gene transcription, and increases in ROS levels. Rotenone, an inhibitor of mitochondrial complex I, abolished the increase in ROS signal, the activation of NF-kappa B, and TNF-alpha gene transcription during hypoxia. LPS stimulated NF-kappa B and TNF-alpha gene transcription but not ROS generation in J774.1 cells. Rotenone, pyrrolidinedithiocarbamic acid, and N-acetylcysteine had no effect on the LPS stimulation of NF-kappa B and TNF-alpha gene transcription, indicating that LPS activates NF-kappa B and TNF-alpha gene transcription through a ROS-independent mechanism. These results indicate that mitochondrial ROS are required for the hypoxic activation of NF-kappa B and TNF-alpha gene transcription, but not for the LPS activation of NF-kappa B.

Animals↗
PubMed2000

Outer mitochondrial membrane permeability can regulate coupled respiration and cell survival.

Coupled cellular respiration requires that ATP and ADP be efficiently exchanged between the cytosol and the mitochondrial matrix. When growth factors are withdrawn from dependent cells, metabolism is disrupted by a defect in ATP/ADP exchange across the mitochondrial membranes. Unexpectedly, we find that this defect results from loss of outer mitochondrial membrane permeability to metabolic anions. This decrease in anion permeability correlates with the changes in conductance properties that accompany closure of the voltage-dependent anion channel (also known as mitochondrial porin). Loss of outer membrane permeability (i) results in the accumulation of stored metabolic energy within the intermembrane space in the form of creatine phosphate, (ii) is prevented by the outer mitochondrial membrane proteins Bcl-x(L) and Bcl-2, and (iii) can be reversed by growth factor readdition. If outer membrane impermeability persists, the disruption of mitochondrial homeostasis culminates in loss of outer mitochondrial membrane integrity, cytochrome c redistribution, and apoptosis. The recognition that outer membrane permeability is regulated under physiological conditions has important implications for the understanding of bioenergetics and cell survival.

Adenosine Diphosphate↗
PubMed2000

Cellular oxygen sensing by mitochondria: old questions, new insight.

Hypoxia elicits a variety of adaptive responses at the tissue level, at the cellular level, and at the molecular level. A physiological response to hypoxia requires the existence of an O(2) sensor coupled to a signal transduction system, which in turn activates the functional response. Although much has been learned about the signaling systems activated by hypoxia, no consensus exists regarding the nature of the underlying O(2) sensor or whether multiple sensors exist. Among previously considered mechanisms, heme proteins have been suggested to undergo allosteric modification in response to O(2) binding or release at different PO(2) levels. Other studies suggest that ion channels may change conductance as a function of PO(2), allowing them to signal the onset of hypoxia. Still other studies suggest that NADPH oxidase may decrease its generation of reactive O(2) species (ROS) during hypoxia. Recent data suggest that mitochondria may function as O(2) sensors by increasing their generation of ROS during hypoxia. These oxidant signals appear to act as second messengers in the adaptive responses to hypoxia in a variety of cell types. Such observations contribute to a growing awareness that mitochondria do more than just generate ATP, in that they initiate signaling cascades involved in adaptive responses to hypoxia and that they participate in the control of cell death pathways.

Animals↗
PubMed1999

Cells depleted of mitochondrial DNA (rho0) yield insight into physiological mechanisms.

A resurgence of interest in mitochondrial physiology has recently developed as a result of new experimental data demonstrating that mitochondria function as important participants in a diverse collection of novel intracellular signaling pathways. Cells depleted of mitochondrial DNA, or rho0 cells, lack critical respiratory chain catalytic subunits that are encoded in the mitochondrial genome. Although rho0 cells contain petit mitochondria, they cannot support normal oxidative phosphorylation and must survive and replicate using ATP derived solely from glycolysis. Without a functional electron transport chain, rho0 cells cannot normally regulate redox potential and their mitochondria appear to be incapable of generating reactive oxygen species. Emerging evidence suggests that these signals are important components in a number of mitochondria-initiated signaling pathways. The present article focuses on how rho0 cells have contributed to an understanding of the role that mitochondria play in distinct physiological pathways involved with apoptosis, glucose-induced insulin secretion, and oxygen sensing.

Animals↗
PubMed1999

Bcl-xL prevents cell death following growth factor withdrawal by facilitating mitochondrial ATP/ADP exchange.

Growth factor withdrawal is associated with a metabolic arrest that can result in apoptosis. Cell death is preceded by loss of outer mitochondrial membrane integrity and cytochrome c release. These mitochondrial events appear to follow a relative increase in mitochondrial membrane potential. This change in membrane potential results from the failure of the adenine nucleotide translocator (ANT)/voltage-dependent anion channel (VDAC) complex to maintain ATP/ADP exchange. Bcl-xL expression allows growth factor-deprived cells to maintain sufficient mitochondrial ATP/ADP exchange to sustain coupled respiration. These data demonstrate that mitochondrial adenylate transport is under active regulation. Efficient exchange of ADP for ATP is promoted by Bcl-xL expression permitting oxidative phosphorylation to be regulated by cellular ATP/ADP levels and allowing mitochondria to adapt to changes in metabolic demand.

Adenosine Diphosphate↗
PubMed1999

Endothelial permeability and IL-6 production during hypoxia: role of ROS in signal transduction.

Prolonged hypoxia produces reversible changes in endothelial permeability, but the mechanisms involved are not fully known. Previous studies have implicated reactive oxygen species (ROS) and cytokines in the regulation of permeability. We tested whether prolonged hypoxia alters permeability to increasing ROS generation, which amplifies cytokine production. Human umbilical vein endothelial cell (HUVEC) monolayers were exposed to hypoxia while secretion of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1alpha, IL-6, and IL-8 was measured. IL-6 and IL-8 secretion increased fourfold over 24 h in a pattern corresponding to changes in HUVEC permeability measured by transendothelial electrical resistance (TEER). Addition of exogenous IL-6 to normoxic HUVEC monolayers caused time-dependent changes in TEER that mimicked the hypoxic response. An antibody to IL-6 significantly attenuated the hypoxia-induced changes in TEER (86 +/- 4 vs. 63 +/- 3% with hypoxia alone at 18 h), whereas treatment with anti-IL-8 had no effect. To determine the role of hypoxia-induced ROS on this response, HUVEC monolayers were incubated with the antioxidants ebselen (50 microM) and N-acetyl-L-cysteine (NAC, 1 mM) before hypoxia. Antioxidants attenuated hypoxia-induced IL-6 secretion (13 +/- 2 pg/ml with ebselen and 19 +/- 3 pg/ml with NAC vs. 140 +/- 15 pg/ml with hypoxia). Ebselen and NAC prevented changes in TEER during hypoxia (94 +/- 2% with ebselen and 90 +/- 6% with NAC vs. 63 +/- 3% with hypoxia at 18 h). N-nitro-L-arginine (500 microM) did not decrease hypoxia-induced changes in dichlorofluorescin fluorescence, IL-6 secretion, or TEER. Thus ROS generated during hypoxia act as signaling elements, regulating secretion of the proinflammatory cytokines that lead to alterations of endothelial permeability.

Antibodies↗
PubMed1998

Mitochondrial reactive oxygen species trigger hypoxia-induced transcription.

Transcriptional activation of erythropoietin, glycolytic enzymes, and vascular endothelial growth factor occurs during hypoxia or in response to cobalt chloride (CoCl2) in Hep3B cells. However, neither the mechanism of cellular O2 sensing nor that of cobalt is fully understood. We tested whether mitochondria act as O2 sensors during hypoxia and whether hypoxia and cobalt activate transcription by increasing generation of reactive oxygen species (ROS). Results show (i) wild-type Hep3B cells increase ROS generation during hypoxia (1. 5% O2) or CoCl2 incubation, (ii) Hep3B cells depleted of mitochondrial DNA (rho0 cells) fail to respire, fail to activate mRNA for erythropoietin, glycolytic enzymes, or vascular endothelial growth factor during hypoxia, and fail to increase ROS generation during hypoxia; (iii) rho0 cells increase ROS generation in response to CoCl2 and retain the ability to induce expression of these genes; and (iv) the antioxidants pyrrolidine dithiocarbamate and ebselen abolish transcriptional activation of these genes during hypoxia or CoCl2 in wild-type cells, and abolish the response to CoCl2 in rho degrees cells. Thus, hypoxia activates transcription via a mitochondria-dependent signaling process involving increased ROS, whereas CoCl2 activates transcription by stimulating ROS generation via a mitochondria-independent mechanism.

Adenosine Triphosphate↗
PubMed1998

Intracellular signaling by reactive oxygen species during hypoxia in cardiomyocytes.

Cardiomyocytes suppress contraction and O2 consumption during hypoxia. Cytochrome oxidase undergoes a decrease in Vmax during hypoxia, which could alter mitochondrial redox and increase generation of reactive oxygen species (ROS). We therefore tested whether ROS generated by mitochondria act as second messengers in the signaling pathway linking the detection of O2 with the functional response. Contracting cardiomyocytes were superfused under controlled O2 conditions while fluorescence imaging of 2, 7-dichlorofluorescein (DCF) was used to assess ROS generation. Compared with normoxia (PO2 approximately 107 torr, 15% O2), graded increases in DCF fluorescence were seen during hypoxia, with responses at PO2 = 7 torr > 20 torr > 35 torr. The antioxidants 2-mercaptopropionyl glycine and 1,10-phenanthroline attenuated these increases and abolished the inhibition of contraction. Superfusion of normoxic cells with H2O2 (25 microM) for >60 min mimicked the effects of hypoxia by eliciting decreases in contraction that were reversible after washout of H2O2. To test the role of cytochrome oxidase, sodium azide (0.75-2 microM) was added during normoxia to reduce the Vmax of the enzyme. Azide produced graded increases in ROS signaling, accompanied by graded decreases in contraction that were reversible. These results demonstrate that mitochondria respond to graded hypoxia by increasing the generation of ROS and suggest that cytochrome oxidase may contribute to this O2 sensing.

Animals↗
PubMed1998

Hibernation during hypoxia in cardiomyocytes. Role of mitochondria as the O2 sensor.

During myocardial hibernation, decreases in coronary perfusion elicit inhibition of contraction, suggesting that energy demand is attenuated. We previously found an inhibition of contraction and O2 consumption during hypoxia (3% O2; PO2 = 20 torr for >2 h) in cardiomyocytes, which was reversible after reoxygenation. This study sought to determine whether mitochondria function as cellular O2 sensors mediating this response. Embryonic cardiomyocytes were studied under controlled O2 conditions. Hypoxia produced no acute decrease in mitochondrial potential as assessed using tetramethylrhodamine ethylester (TMRE). Cellular [ATP] was preserved throughout hypoxia, as assessed using the probe Magnesium Green. Thus, ATP synthesis and utilization remained closely coupled. Cells adapted to hypoxia for >2 h exhibited a 4% increase in mitochondrial potential upon reoxygenation, suggesting that a partial inhibition of cytochrome c oxidase had existed. To test whether the oxidase serves as an O2 sensor, azide was administered (1 mM) to simulate the effects of hypoxia by lowering the Vmax of the oxidase. The effects of azide on contraction and mitochondrial potential mimicked the response to hypoxia. We conclude that partial inhibition of cytochrome oxidase during hypoxia allows mitochondria to function as the O2 sensor mediating the decreases in ATP utilization and O2 consumption during hypoxia.

Adenosine Triphosphate↗