Biomedical subjects

Judith Haendeler

Publications and source records attributed to Judith Haendeler.

32 records · Page 2Linked to original sources

PubMed2003

Thioredoxin: a key regulator of cardiovascular homeostasis.

The thioredoxin (TRX) system (TRX, TRX reductase, and NADPH) is a ubiquitous thiol oxidoreductase system that regulates cellular reduction/oxidation (redox) status. The oxidation mechanism for disease pathogenesis states that an imbalance in cell redox state alters function of multiple cell pathways. In this study, we review the essential role for TRX to limit oxidative stress directly via antioxidant effects and indirectly by protein-protein interaction with key signaling molecules, such as apoptosis signal-regulating kinase 1. We propose that TRX and its endogenous regulators are important future targets to develop clinical therapies for cardiovascular disorders associated with oxidative stress.

Animals↗

PubMed2003

GIT1 mediates Src-dependent activation of phospholipase Cgamma by angiotensin II and epidermal growth factor.

Critical events for vasoconstrictor and growth factor signal transduction include stimulation of phospholipase Cgamma (PLCgamma) and elevation of intracellular calcium. c-Src has been proposed as a common mediator for these signals activated by both G protein-coupled receptors (GPCRs) and tyrosine kinase-coupled receptors (TKRs). Here we show that the GPCR kinase-interacting protein-1 (GIT1) is a substrate for c-Src that undergoes tyrosine phosphorylation in response to angiotensin II (AngII) and EGF in vascular smooth muscle and 293 cells. GIT1 associates with PLCgamma via the PLCgamma Src homology 2 and 3 domains constitutively, and the interaction is unaltered by AngII and EGF. GIT1 interaction with PLCgamma is required for PLCgamma activation based on inhibition of tyrosine phosphorylation and calcium mobilization after GIT1 knockdown with antisense GIT1 oligonucleotides. GIT1 interacts with PLCgamma via a novel Spa homology domain (SHD) and a coiled-coil domain. Deletion mutation analysis showed that GIT1(SHD) is required for AngII- and EGF-mediated PLCgamma activation (measured by phosphorylation of Tyr783 and inositol 1,4,5-trisphosphate formation). We propose that GIT1 is a novel regulator of PLCgamma function that mediates PLCgamma activation by c-Src and integrates signal transduction by GPCRs and TKRs.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Shear stress increases the amount of S-nitrosylated molecules in endothelial cells: important role for signal transduction.

Laminar flow (shear stress) is an important stimulus for nitric oxide (NO) synthesis in endothelial cells. NO can react with free SH-groups of different proteins leading to S-nitrosylation. Since S-nitrosylation of proteins is an important regulator of protein functions, we investigated the effect of endogenously synthesized NO. Exposure to shear stress significantly increased the overall S-nitrosylation of proteins in endothelial cells. Interestingly, shear stress increased S-nitrosylation of specific target proteins, i.e. the catalytic p17 subunit of caspase-3, the GTPase p21ras and the oxidoreductase thioredoxin. S-nitrosylation resulted in an inhibition of caspase-3 and in an augmented activity of p21ras and thioredoxin. These data suggest that long term exposure to shear stress exerts its different atheroprotective effects at least in part via increased S-nitrosylation of specific signaling proteins.

Caspase 3↗

PubMed2003

HMG-CoA reductase inhibitors reduce senescence and increase proliferation of endothelial progenitor cells via regulation of cell cycle regulatory genes.

Endothelial progenitor cells (EPCs) play an important role in postnatal neovascularization of ischemic tissue. Ex vivo expansion of EPCs might be useful for potential clinical cell therapy of myocardial ischemia. However, cultivation of primary cells leads to cellular aging (senescence), thereby severely limiting the proliferative capacity. Therefore, we investigated whether statins might be able to prevent senescence of EPCs. EPCs were isolated from peripheral blood and characterized. After ex vivo cultivation, EPCs became senescent as determined by acidic beta-galactosidase staining. Atorvastatin or mevastatin dose-dependently inhibited the onset of EPC senescence in culture. Moreover, atorvastatin increased proliferation of EPCs as assessed by BrdU incorporation and colony-forming capacity. Whereas geranylgeranylpyrophosphate or farnesylpyrophosphate reduced the senescence inhibitory effect of atorvastatin, NO synthase inhibition, antioxidants, or Rho kinase inhibitors had no effect. To get further insights into the underlying downstream effects of statins, we measured telomerase activity and determined the expression of various cell cycle regulatory genes by using a microarray assay. Whereas telomerase activity did not change, atorvastatin modulated expression of cell cycle genes including upregulation of cyclins and downregulation of the cell cycle inhibitor p27Kip1. Taken together, statins inhibited senescence of EPCs independent of NO, reactive oxygen species, and Rho kinase, but dependent on geranylgeranylpyrophosphate. Atorvastatin-mediated prevention of EPC senescence appears to be mediated by the regulation of various cell cycle proteins. The inhibition of EPC senescence and induction of EPC proliferation by statins in vitro may importantly improve the functional activity of EPCs for potential cell therapy.

Atorvastatin↗

PubMed2003

Regulation of telomerase activity and anti-apoptotic function by protein-protein interaction and phosphorylation.

The enzyme telomerase is necessary for the synthesis and maintenance of telomere length. The catalytic subunit, telomerase reverse transcriptase (TERT), is regulated by interaction with the 90 kDa heat shock protein (HSP90) and by Akt-dependent phosphorylation. Here, we demonstrate that HSP90 and Akt physically interact with TERT. Treatment of cells with novobiocin, which blocks C-terminal interaction of HSP90, disrupted HSP90 binding to Akt, induced Akt dephosphorylation and significantly reduced telomerase activity. The reduction of TERT activity by novobiocin was associated with an increase in apoptosis. Likewise, the induction of Akt dephosphorylation by protein phosphatase 2A (PP2A) reduced telomerase activity. HSP90 is known to prevent PP2A-mediated dephosphorylation of Akt. To investigate whether the effect of novobiocin is due to the reduction of Akt or TERT phosphorylation, we overexpressed a phospho-mimetic, active Akt (T308D/S473D). Akt (T308D/S473D) prevented novobiocin-induced reduction of telomerase activity and the stimulation of apoptosis. Moreover, overexpression of a dominant negative PP2A construct (PP2A(L199P)) as well as incubation with the PP2A inhibitor okadaic acid blocked the inhibition of telomerase activity by novobiocin. These data suggest that the association between HSP90, Akt and TERT in concert with the phosphorylation of TERT is necessary for maintaining telomerase activity and inhibition of apoptosis.

Apoptosis↗

PubMed2003

Hydrogen peroxide triggers nuclear export of telomerase reverse transcriptase via Src kinase family-dependent phosphorylation of tyrosine 707.

The regulation of telomerase reverse transcriptase (TERT) plays an important role in the proliferative capacity and survival of cells. Here, we report that exogenously as well as endogenously induced oxidative stress leads to translocation of endogenous as well as overexpressed human TERT from the nucleus into the cytosol. TERT is transported through the nuclear pores in a leptomycin-sensitive and Ran GTPase-dependent process. H(2)O(2)-induced nuclear export of TERT is preceded by TERT tyrosine phosphorylation at position 707 and prevented by the Src kinase family inhibitor PP1. Oxidative stress-induced nuclear export of TERT depends on association with the Ran GTPase. In contrast, mutation of tyrosine 707 inhibits phosphorylation induced by oxidative stress and prevents association with Ran and nuclear export of TERT. Moreover, inhibition of tyrosine phosphorylation at 707 increases the antiapoptotic capacity of TERT. Taken together, depletion of nuclear TERT by tyrosine phosphorylation-dependent nuclear export of TERT is a novel mechanism for regulation of TERT localization, which reduces the antiapoptotic activity of TERT.

Active Transport, Cell Nucleus↗

PubMed2002

Fluid shear stress attenuates hydrogen peroxide-induced c-Jun NH2-terminal kinase activation via a glutathione reductase-mediated mechanism.

c-Jun NH2-terminal kinase (JNK) is activated by a number of cellular stimuli including reactive oxygen species (ROS). Previous studies have demonstrated that fluid shear stress (flow) inhibits cytokine-induced JNK activation in endothelial cells (ECs). In the present study, we show JNK activation by ROS in ECs and hypothesized that flow inhibits ROS-induced JNK activation in ECs via modulation of cellular protection systems against ROS. JNK was activated by 300 micro mol/L hydrogen peroxide (H2O2) in bovine lung microvascular ECs (BLMVECs) with a peak at 60 minutes after stimulation (6.3+/-1.2-fold increase). Preexposure of BLMVECs to physiological steady laminar flow (shear stress=12 dyne/cm2) for 10 minutes significantly decreased H2O2-induced JNK activation. Thioredoxin and glutathione are cellular antioxidants that protect cells against ROS. Flow induced a significant increase in the ratio of reduced glutathione to oxidized glutathione consistent with a 1.6-fold increase in glutathione reductase (GR) activity. Preincubation of BLMVECs with the GR inhibitor, 1,3 bis-(2 chloroethyl)-1-nitrosourea, abolished the inhibitory effect of flow. In contrast, preincubation of BLMVECs with azelaic acid, a specific inhibitor for thioredoxin reductase, did not alter the effect of flow on H2O2-induced JNK activation. Overexpression of GR mimicked the effect of flow to inhibit JNK activation. These results suggest that flow activates GR, an important regulator of the intracellular redox state of glutathione, and exerts a protective mechanism against oxidative stress in endothelial cells.

Animals↗

PubMed2002

Hypoxic induction of the hypoxia-inducible factor is mediated via the adaptor protein Shc in endothelial cells.

Tyrosine kinase cascades may play a role in the hypoxic regulation of hypoxia-inducible factor (HIF)-1. We investigated the role of tyrosine kinase phosphorylation and of the Shc/Ras cascade on hypoxic HIF-1 stabilization. Exposure of human umbilical vein endothelial cells to hypoxia results in HIF protein stabilization as early as 10 minutes, with a maximum at 3 hours, and also in Shc tyrosine phosphorylation, with a maximum at 10 minutes. To test whether Shc directly mediates hypoxia-induced HIF stabilization, human umbilical vein endothelial cells were transfected with a dominant-negative Shc mutant (dnShc), resulting in significantly reduced HIF protein levels compared with control. Similar results were obtained with cells transfected with dominant-negative Ras, a known downstream effector of Shc. Hypoxia-induced Ras activity was significantly reduced in cells transfected with dnShc compared with control levels, indicating that Ras indeed acts downstream from Shc. Moreover, cells pretreated with a specific Raf-1 kinase inhibitor, a known downstream effector of Ras, exhibited reduced HIF protein levels. To examine the functional consequences of Shc in hypoxic signaling, HIF-1 ubiquitination, protein stabilization, and endothelial cell migration were assessed. Overexpression of dnShc increased ubiquitination of HIF-1 and reduced the half-life of the protein. Moreover, dnShc, dominant-negative Ras, or the Raf-1 kinase inhibitor significantly inhibited migration under hypoxia. Thus, Shc in concert with Ras and Raf-1 contributes to hypoxia-induced HIF-1alpha protein stabilization and endothelial cell migration.

Adaptor Proteins, Signal Transducing↗

PubMed2002

The vascular NADPH oxidase subunit p47phox is involved in redox-mediated gene expression.

An NADPH oxidase is thought to be a main source of vascular superoxide (O(2)(-)) production. The functional role of this oxidase, however, and the contribution of the different subunits of the enzyme to cellular signaling are still incompletely understood. We determined the role of the p47phox subunit of the oxidase in O(2)(-) generation and signaling in aortic rings and cultured smooth muscle cells (SMC) from wild-type (WT) and p47phox-deficient (p47phox -/-) mice. Basal O(2)(-) levels in aortae of p47phox -/- mice were lower than those in WT aortae. Infusion of [val(5)]-angiotensin II increased O(2)(-) levels in aortae from WT more than in aortae from p47phox -/- mice. O(2)(-) generation was similar in quiescent SMC from WT and p47phox -/- mice. However, exposure to thrombin selectively increased O(2)(-) generation in VSMC from WT, but not from p47phox -/- mice. Thrombin-activated redox-mediated signal transduction and gene expression was attenuated in VSMC from p47phox -/- compared to cells from WT mice as determined by p38 MAP kinase activation and VEGF gene expression. We conclude that p47phox is important for vascular ROS production and redox-modulated signaling and gene expression in VSMC.

Angiotensin II↗

PubMed2002

Dephosphorylation of endothelial nitric oxide synthase contributes to the anti-angiogenic effects of endostatin.

Endostatin is an anti-angiogenic factor that inhibits endothelial cell (EC) migration and induces EC apoptosis. Because nitric oxide (NO) plays a key role in vascular endothelial growth factor (VEGF)-induced angiogenesis, we hypothesized that endostatin interferes with the activation of the endothelial NO synthase (eNOS). Human recombinant endostatin significantly reduced VEGF-induced NO-release, which suggests that endostatin inhibits eNOS activation. Because the activation of eNOS by VEGF is associated with the Akt-dependent phosphorylation of eNOS at Ser1177, we investigated whether endostatin interferes with phosphorylation of eNOS. Endostatin reduced VEGF-induced phosphorylation of eNOS at Ser1177, whereas Akt phosphorylation was not affected. Coinciding with the inhibition of eNOS phosphorylation, endostatin completely blocked VEGF-induced EC migration. The NO-donor SNAP reversed the inhibitory effect of endostatin on EC migration. In addition, endostatin significantly inhibited VEGF-induced tube formation, whereas endostatin did not affect tube formation induced by NO. Finally, a non-dephosphorylatable constitutive active eNOS construct (S1177D), but not constitutive active Akt, abolished the inhibitory effect of endostatin on EC migration. Endostatin activated PP2A, which is known to directly dephosphorylate eNOS at Ser1177. Inhibition of PP2A prevented the inhibitory effect of endostatin. Thus, endostatin inhibits VEGF-induced EC migration and angiogenesis upstream of NO-synthesis via dephosphorylation of eNOS at Ser1177.

Angiogenesis Inhibitors↗

PubMed2002

Angiotensin II-induced upregulation of MAP kinase phosphatase-3 mRNA levels mediates endothelial cell apoptosis.

Angiotensin II (Ang II) is central to the pathobiology of atherosclerosis. In endothelial cells (EC), Ang II induces apoptosis. The MAP kinase ERK1/2 plays a key role in regulating cell survival. We therefore investigated the effect of Ang II on ERK1/2. Incubation of EC with Ang II led to the dephosphorylation of ERK1/2 (43% of control). To characterize the phosphatase involved, we investigated the effect of Ang II on MAP kinase phosphatase expression. Ang II induced MAP kinase phosphatase-3 (MKP-3) mRNA levels to about 2-fold, whereas MKP-1 expression was not affected. Transfection with a dominant negative MKP-3 construct (dnMKP-3mt) prevented the Ang II-induced ERK1/2 dephosphorylation and apoptosis in EC (p < 0.001). ERK1/2 inactivation has been shown to result in the dephosphorylation and proteasomal degradation of the antiapoptotic protein Bcl-2. Ang II induced the degradation of Bcl-2 wild type, whereas the dephosphorylation-resistant Bcl-2 construct mimicking phosphorylation by ERK1/2 was resistant to Ang II stimulation. These results indicate that Ang II-induced apoptosis signaling in human EC is mediated via MKP-3-dependent dephosphorylation of ERK1/2, which in turn leads to the degradation of Bcl-2.

Angiotensin II↗

PubMed2002

Redox regulatory and anti-apoptotic functions of thioredoxin depend on S-nitrosylation at cysteine 69.

Thioredoxin 1 (Trx) is a known redox regulator that is implicated in the redox control of cell growth and apoptosis inhibition. Here we show that Trx is essential for maintaining the content of S-nitrosylated molecules in endothelial cells. Trx itself is S-nitrosylated at cysteine 69 under basal conditions, and this S-nitrosylation is required for scavenging reactive oxygen species and for preserving the redox regulatory activity of Trx. S-nitrosylation of Trx also contributes to the anti-apoptotic function of Trx. Thus, Trx can exert its complete redox regulatory and anti-apoptotic functions in endothelial cells only when cysteine 69 is S-nitrosylated.

Amino Acid Sequence↗

PubMed2002

Regulation of endothelial cell apoptosis in atherothrombosis.

Increasing evidence suggests that endothelial cell apoptosis might contribute to atherogenesis, plaque erosion and acute coronary syndromes. The present review article summarizes the current findings and discusses the potential role of endothelial cell apoptosis for coronary artery disease progression. Moreover, the signalling events that critically regulate endothelial cell apoptosis are discussed. The article focuses on the regulation of endothelial cell apoptosis by the redox balance, which is mainly determined by reactive oxygen species and nitric oxide. Moreover, the role of S-nitrosylation of protein targets for protection of endothelial cell apoptosis will be discussed.

Animals↗

PubMedDate not supplied

Thioredoxin-1 and posttranslational modifications.

Thioredoxin-1 is a 12 kDa protein that consists of a redox regulatory domain containing the active cysteine residues 32 and 35. These cysteines are conserved from bacteria to human. Unlike thioredoxins from lower species, mammalian thioredoxin-1 contains three additional nonactive cysteine residues at positions 62, 69, and 73 (for human thioredoxin-1). Key biological functions of thioredoxin-1 are antioxidative, anti-apoptotic, and pro-proliferative properties. Thioredoxin-1 is regulated by the ability of the thioredoxin reductase to reduce oxidized thioredoxin-1 at cysteines 32 and 35. However, posttranslational modifications of thioredoxin-1, including glutathionylation, thiol-oxidation, and S-nitros(yl)ation, at the nonactive cysteines importantly contribute to the regulation and functions of thioredoxin-1. This review focuses on the posttranslational modifications of the active and nonactive cysteines and their contribution for functional regulation of thioredoxin-1.

Animals↗