104 results on '"Lassègue B"'
Search Results
2. The C242T CYBA Polymorphism as a Major Determinant of NADPH Oxidase Activity in Patients with Cardiovascular Disease
- Author
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Mehranpour, P., Wang, S. S., Blanco, R. R., Li, W., Song, Q., Lassègue, B., Dikalov, S. I., Austin, H., and Zafari, A. M.
- Published
- 2009
3. Prolonged exposure to agonist results in a reduction in the levels of the Gq/G11 alpha subunits in cultured vascular smooth muscle cells
- Author
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Kai H, Fukui T, Lassègue B, Shah A, Ca, Minieri, and Kathy Griendling
- Subjects
Male ,Base Sequence ,Angiotensin II ,Molecular Sequence Data ,Muscle, Smooth, Vascular ,Rats ,Enzyme Activation ,Rats, Sprague-Dawley ,Kinetics ,GTP-Binding Proteins ,Animals ,Tetradecanoylphorbol Acetate ,RNA, Messenger ,Cells, Cultured ,Protein Kinase C ,DNA Primers ,Signal Transduction - Abstract
Recent studies have shown that G proteins are a potential regulatory site in the transmembrane signaling cascade. The aim of this study was to examine the effects of prolonged agonist exposure on expression of the Gq class of G protein alpha subunits (G alpha q/G alpha 11) in cultured rat vascular smooth muscle cells (VSMC). Treatment with 100 nM angiotensin II (Ang II) led to a substantial sustained down-regulation of cellular levels of immunologically detectable G alpha q/G alpha 11 by 50% within 6 hr. The effect of Ang II was dose dependent with an EC50 of 2 nM and was specifically blocked by the vascular type-1 Ang II receptor-specific antagonist losartan. The Ang II-induced reduction in cellular levels of G protein alpha subunits was specific for G alpha q/G alpha 11. The calcium ionophore ionomycin or activators of ubiquitous protein kinases (phorbol-12-myristate-13-acetate, forskolin, and 8-bromo-cGMP) did not mimic the effects of Ang II. However, [Arg8]vasopressin also induced a significant loss in cellular G alpha q/G alpha 11 levels. Ang II-induced G alpha q/G alpha 11 down-regulation was reversed by prevention of cellular receptor processing with phenylarsine oxide or chronic potassium depletion. The effects of Ang II on G alpha q/G alpha 11 levels were inhibited when protein kinase C activity was abolished. G alpha q mRNA levels were down-regulated by 30% after 4-hr incubation with Ang II, in part by transcriptional regulation. Although a short term vasopressin pretreatment had no effect on inositol-1,4,5-trisphosphate (IP3) generation in response to subsequent Ang II stimulation, a partial heterologous desensitization of the IP3 response was induced after a long term vasopressin pretreatment, which concurrently down-regulated cellular G alpha q/G alpha 11 levels. Homologous desensitization of IP3 generation on a second Ang II stimulation was observed after both a short and long term Ang II pretreatment. In conclusion, prolonged exposure to Ang II induces down-regulation of cellular G alpha q/G alpha 11 levels in intact VSMC. The effect of Ang II appears to be mediated by the signaling pathway sensitive to inhibition of receptor processing. The present study raises the possibility that agonist-induced G alpha q/G alpha 11 down-regulation participates in the mechanism of long term desensitization of the G alpha q/G alpha 11-mediated signaling system in VSMC.
- Published
- 1996
4. Agonist-induced phosphorylation of the vascular type 1 angiotensin II receptor.
- Author
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Kai, H, primary, Griendling, K K, additional, Lassègue, B, additional, Ollerenshaw, J D, additional, Runge, M S, additional, and Alexander, R W, additional
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- 1994
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5. Angiotensin II stimulates phosphorylation of high-molecular-mass cytosolic phospholipase A2 in vascular smooth-muscle cells
- Author
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Rao, G N, primary, Lassègue, B, additional, Alexander, R W, additional, and Griendling, K K, additional
- Published
- 1994
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6. Phosphatidylcholine is a major source of phosphatidic acid and diacylglycerol in angiotensin II-stimulated vascular smooth-muscle cells
- Author
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Lassègue, B, primary, Alexander, R W, additional, Clark, M, additional, Akers, M, additional, and Griendling, K K, additional
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- 1993
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7. Angiotensin II-induced phosphatidylcholine hydrolysis in cultured vascular smooth-muscle cells. Regulation and localization
- Author
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Lassègue, B, primary, Alexander, R W, additional, Clark, M, additional, and Griendling, K K, additional
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- 1991
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8. Nox1 overexpression potentiates angiotensin II-induced hypertension and vascular smooth muscle hypertrophy in transgenic mice.
- Author
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Dikalova A, Clempus R, Lassègue B, Cheng G, McCoy J, Dikalov S, San Martin A, Lyle A, Weber DS, Weiss D, Taylor WR, Schmidt HH, Owens GK, Lambeth JD, and Griendling KK
- Published
- 2005
9. Angiotensin II type 1 receptor: relationship with caveolae and caveolin after initial agonist stimulation.
- Author
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Ishizaka, Nobukazu, Griendling, Kathy K., Lassegue, Bernard, Alexander, R. Wayne, Ishizaka, N, Griendling, K K, Lassègue, B, and Alexander, R W
- Published
- 1998
10. Angiotensin II down-regulates the vascular smooth muscle AT1 receptor by transcriptional and post-transcriptional mechanisms: evidence for homologous and heterologous regulation.
- Author
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Lassègue, B, Alexander, R W, Nickenig, G, Clark, M, Murphy, T J, and Griendling, K K
- Abstract
The vascular angiotensin II (ANG II) receptor (AT1) is a central component of the renin-angiotensin system; thus, regulation of its expression is likely to be important in cardiovascular responsiveness. We demonstrate that ANG II down-regulates its receptor in rat aortic vascular smooth muscle cells. Incubation for 4 hr with 100 nM ANG II decreased AT1 mRNA and protein by 70% and 35%, respectively. This homologous down-regulation was concentration and time dependent and was blocked by the AT1 antagonist losartan. It did not appear to be mediated by protein kinase C or other protein kinases but was dependent on the sustained signaling pathway sensitive to phenylarsine oxide. Heterologous down-regulation was observed with the agonists alpha-thrombin and ATP and the cAMP-increasing agent forskolin. ANG II inhibited transcription by 50% and destabilized the AT1 mRNA. Down-regulation of AT1 mRNA was blocked by transcription and translation inhibitors, suggesting that it required expression of a protein factor or factors. These results indicate that ANG II down-regulates its vascular receptor by both transcriptional and post-transcriptional mechanisms. Homologous and heterologous down-regulation of the AT1 receptor may participate in the coordinated physiological adaptation of vascular tone to vasoactive hormones.
- Published
- 1995
11. Hydrogen peroxide stimulates transcription of c-jun in vascular smooth muscle cells: role of arachidonic acid
- Author
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Gn, Rao, Lassègue B, Kathy Griendling, and Rw, Alexander
- Subjects
Male ,Arachidonic Acid ,Transcription, Genetic ,Proto-Oncogene Proteins c-jun ,Down-Regulation ,Glyceraldehyde-3-Phosphate Dehydrogenases ,Hydrogen Peroxide ,Muscle, Smooth, Vascular ,Peptide Fragments ,Phospholipases A ,Rats ,Rats, Sprague-Dawley ,Phospholipases A2 ,Quinacrine ,Animals ,Masoprocol ,RNA, Messenger ,Cells, Cultured ,Protein Kinase C - Abstract
We reported previously that hydrogen peroxide induces DNA synthesis in rat aortic smooth muscle (RASM) cells. In the present paper we studied the mechanism by which hydrogen peroxide induces c-jun mRNA, an early response gene whose activation is required for mitogen-stimulated cell growth. Hydrogen peroxide induced c-jun mRNA in growth-arrested RASM cells in a time dependent manner. This stimulation was significantly inhibited by mepacrine, a phospholipase A2 (PLA2) inhibitor. Arachidonic acid, a PLA2 product, also increased c-jun mRNA with a time course similar to that of hydrogen peroxide. The increases in c-jun mRNA induced by hydrogen peroxide and arachidonic acid were significantly reduced (55%) by down-regulation of protein kinase C with a phorbol ester. Furthermore, the effect of hydrogen peroxide on c-jun mRNA was also reduced by NDGA, an inhibitor of the lipoxygenase-cytochrome P450 mono-oxygenase system, suggesting that metabolism of arachidonic acid through this pathway is required for the induction of c-jun mRNA by oxidants. Both hydrogen peroxide and arachidonic acid significantly increased c-jun transcription as demonstrated by nuclear run-on assays. Together these observations suggest that: (1) the induction of c-jun mRNA by hydrogen peroxide is mediated by PLA2-dependent arachidonic acid release and metabolism through the lipoxygenase-cytochrome P450 mono-oxygenase system; (2) PKC appears to be involved in this signaling pathway and (3) the induction of c-jun mRNA by hydrogen peroxide in RASM cells is due to increased transcription.
12. The vascular angiotensin (AT1) receptor
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Kathy Griendling, Lassègue B, and Rw, Alexander
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Receptors, Angiotensin ,Molecular Sequence Data ,Animals ,Blood Vessels ,Humans ,Amino Acid Sequence ,Cloning, Molecular ,Signal Transduction
13. Erratum: (Arteriosclerosis, Thrombosis, and Vascular Biology (2007) 27 (42-48))
- Author
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Clempus, R. E., Sorescu, D., Dikalova, A. E., Pounkova, L., Jo, P., Sorescu, G., Schmidt, H. H. H., Lassègue, B., and Kathy Griendling
14. Phenotypic modulation of the calcium signal induced by agonists in vascular smooth muscle cells
- Author
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Andre, Ph., Lassegue, B., Descombes, J.J., and Stoclet, J.C.
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- 1990
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15. Polymerase delta-interacting protein 2 mediates brain vascular permeability by regulating ROS-mediated ZO-1 phosphorylation and localization at the interendothelial border.
- Author
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Wang K, Qu H, Hu R, Lassègue B, Eaton DC, Song C, Mu J, Griendling KK, and Hernandes MS
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- Animals, Phosphorylation, Mice, Humans, Blood-Brain Barrier metabolism, Brain metabolism, Mice, Knockout, Nuclear Proteins metabolism, Nuclear Proteins genetics, Brain Ischemia metabolism, Endothelial Cells metabolism, Tight Junctions metabolism, Zonula Occludens-1 Protein metabolism, Reactive Oxygen Species metabolism, Capillary Permeability
- Abstract
Background: Polymerase delta-interacting protein 2 (Poldip2) is a novel regulator of vascular permeability that has been shown to be involved in aggravating blood-brain barrier (BBB) disruption following stroke; however, the underlying mechanisms are unknown. While endothelial tight junctions (TJ) are critical mediators of BBB permeability, the effect of Poldip2 on TJ function has not been elucidated yet. Here, we aim to define the mechanism by which Poldip2 mediates BBB disruption, specifically focusing on phosphorylation and stabilization of the TJ integral protein ZO-1., Methods and Results: Cerebral ischemia was induced in endothelial-specific Poldip2 knockout mice and controls. Cerebral vascular permeability was assessed by Evans blue dye extravasation. Endothelial-specific Poldip2 deletion abolished Evans blue dye extravasation after ischemia induction. In vitro permeability assays demonstrated that Poldip2 knockdown suppressed TNF-α-induced endothelial cell (EC) permeability. Immunofluorescence staining showed that Poldip2 depletion prevented TNF-α-induced ZO-1 disruption at interendothelial junctions. Conversely, Poldip2 overexpression increased endothelial permeability, loss of ZO-1 localization at cell-cell junctions and enhanced reactive oxygen species (ROS) production. Treatment with the antioxidant N-acetyl cysteine (NAC) reduced Poldip2-induced ZO-1 disruption at inter interendothelial junctions. Immunoprecipitation studies demonstrated Poldip2 overexpression induced tyrosine phosphorylation of ZO-1, which was prevented by treatment with NAC or MitoTEMPO, a mitochondrial ROS scavenger., Conclusions: These data reveal a novel mitochondrial ROS-driven mechanism by which Poldip2 induces ZO-1 tyrosine phosphorylation and promotes EC permeability following cerebral ischemia., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)
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- 2025
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16. Endothelial Poldip2 regulates sepsis-induced lung injury via Rho pathway activation.
- Author
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Dolmatova EV, Forrester SJ, Wang K, Ou Z, Williams HC, Joseph G, Kumar S, Valdivia A, Kowalczyk AP, Qu H, Jo H, Lassègue B, Hernandes MS, and Griendling KK
- Subjects
- Animals, Endothelium metabolism, Humans, Lung metabolism, Mice, Mitochondrial Proteins genetics, Nuclear Proteins genetics, Tumor Necrosis Factor-alpha metabolism, Lung Injury genetics, Mitochondrial Proteins metabolism, Nuclear Proteins metabolism, Sepsis complications, Sepsis genetics, Sepsis metabolism
- Abstract
Aims: Sepsis-induced lung injury is associated with significant morbidity and mortality. Previously, we showed that heterozygous deletion of polymerase δ-interacting protein 2 (Poldip2) was protective against sepsis-induced lung injury. Since endothelial barrier disruption is thought to be the main mechanism of sepsis-induced lung injury, we sought to determine if the observed protection was specifically due to the effect of reduced endothelial Poldip2., Methods and Results: Endothelial-specific Poldip2 knock-out mice (EC-/-) and their wild-type littermates (EC+/+) were injected with saline or lipopolysaccharide (18 mg/kg) to model sepsis-induced lung injury. At 18 h post-injection mice, were euthanized and bronchoalveolar lavage (BAL) fluid and lung tissue were collected to assess leucocyte infiltration. Poldip2 EC-/- mice showed reduced lung leucocyte infiltration in BAL (0.21 ± 0.9×106 vs. 1.29 ± 1.8×106 cells/mL) and lung tissue (12.7 ± 1.8 vs. 23 ± 3.7% neutrophils of total number of cells) compared to Poldip2 EC+/+ mice. qPCR analysis of the lung tissue revealed a significantly dampened induction of inflammatory gene expression (TNFα 2.23 ± 0.39 vs. 4.15 ± 0.5-fold, IκBα 4.32 ± 1.53 vs. 8.97 ± 1.59-fold), neutrophil chemoattractant gene expression (CXCL1 68.8 ± 29.6 vs. 147 ± 25.7-fold, CXCL2 65 ± 25.6 vs. 215 ± 27.3-fold) and a marker of endothelial activation (VCAM1 1.25 ± 0.25 vs. 3.8 ± 0.38-fold) in Poldip2 EC-/- compared to Poldip2 EC+/+ lungs. An in vitro model using human pulmonary microvascular endothelial cells was used to assess the effect of Poldip2 knock-down on endothelial activation and permeability. TNFα-induced endothelial permeability and VE-cadherin disruption were significantly reduced with siRNA-mediated knock-down of Poldip2 (5 ± 0.5 vs. 17.5 ± 3-fold for permeability, 1.5 ± 0.4 vs. 10.9 ± 1.3-fold for proportion of disrupted VE-cadherin). Poldip2 knock-down altered expression of Rho-GTPase-related genes, which correlated with reduced RhoA activation by TNFα (0.94 ± 0.05 vs. 1.29 ± 0.01 of relative RhoA activity) accompanied by redistribution of active-RhoA staining to the centre of the cell., Conclusion: Poldip2 is a potent regulator of endothelial dysfunction during sepsis-induced lung injury, and its endothelium-specific inhibition may provide clinical benefit., Competing Interests: Conflict of interest: none declared., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)
- Published
- 2022
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17. Myeloid Poldip2 Contributes to the Development of Pulmonary Inflammation by Regulating Neutrophil Adhesion in a Murine Model of Acute Respiratory Distress Syndrome.
- Author
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Ou Z, Dolmatova E, Mandavilli R, Qu H, Gafford G, White T, Valdivia A, Lassègue B, Hernandes MS, and Griendling KK
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- Animals, Cell Adhesion, Disease Models, Animal, Focal Adhesion Kinase 2 metabolism, Integrins metabolism, Lipopolysaccharides pharmacology, Mice, Mice, Inbred C57BL, Mice, Knockout, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Neutrophils metabolism, Neutrophils pathology, Nuclear Proteins genetics, Nuclear Proteins metabolism, Pneumonia genetics, Pneumonia metabolism, Pneumonia pathology, Respiratory Distress Syndrome genetics, Respiratory Distress Syndrome metabolism, Respiratory Distress Syndrome pathology
- Abstract
Background Lung injury, a severe adverse outcome of lipopolysaccharide-induced acute respiratory distress syndrome, is attributed to excessive neutrophil recruitment and effector response. Poldip2 (polymerase δ-interacting protein 2) plays a critical role in regulating endothelial permeability and leukocyte recruitment in acute inflammation. Thus, we hypothesized that myeloid Poldip2 is involved in neutrophil recruitment to inflamed lungs. Methods and Results After characterizing myeloid-specific Poldip2 knockout mice, we showed that at 18 hours post-lipopolysaccharide injection, bronchoalveolar lavage from myeloid Poldip2-deficient mice contained fewer inflammatory cells (8 [4-16] versus 29 [12-57]×10
4 /mL in wild-type mice) and a smaller percentage of neutrophils (30% [28%-34%] versus 38% [33%-41%] in wild-type mice), while the main chemoattractants for neutrophils remained unaffected. In vitro, Poldip2-deficient neutrophils responded as well as wild-type neutrophils to inflammatory stimuli with respect to neutrophil extracellular trap formation, reactive oxygen species production, and induction of cytokines. However, neutrophil adherence to a tumor necrosis factor-α stimulated endothelial monolayer was inhibited by Poldip2 depletion (225 [115-272] wild-type [myePoldip2+/+ ] versus 133 [62-178] myeloid-specific Poldip2 knockout [myePoldip2-/- ] neutrophils) as was transmigration (1.7 [1.3-2.1] versus 1.1 [1.0-1.4] relative to baseline transmigration). To determine the underlying mechanism, we examined the surface expression of β2-integrin, its binding to soluble intercellular adhesion molecule 1, and Pyk2 phosphorylation. Surface expression of β2-integrins was not affected by Poldip2 deletion, whereas β2-integrins and Pyk2 were less activated in Poldip2-deficient neutrophils. Conclusions These results suggest that myeloid Poldip2 is involved in β2-integrin activation during the inflammatory response, which in turn mediates neutrophil-to-endothelium adhesion in lipopolysaccharide-induced acute respiratory distress syndrome.- Published
- 2022
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18. Characterization of Poldip2 knockout mice: Avoiding incorrect gene targeting.
- Author
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Lassègue B, Kumar S, Mandavilli R, Wang K, Tsai M, Kang DW, Demos C, Hernandes MS, San Martín A, Taylor WR, Jo H, and Griendling KK
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- Animals, Membrane Proteins metabolism, Mice, Mice, Knockout, Mitochondrial Proteins metabolism, Nuclear Proteins metabolism, CRISPR-Cas Systems, Gene Targeting, Membrane Proteins genetics, Mitochondrial Proteins deficiency, Mouse Embryonic Stem Cells metabolism, Nuclear Proteins deficiency, RNA-Seq
- Abstract
POLDIP2 is a multifunctional protein whose roles are only partially understood. Our laboratory previously reported physiological studies performed using a mouse gene trap model, which suffered from three limitations: perinatal lethality in homozygotes, constitutive Poldip2 inactivation and inadvertent downregulation of the adjacent Tmem199 gene. To overcome these limitations, we developed a new conditional floxed Poldip2 model. The first part of the present study shows that our initial floxed mice were affected by an unexpected mutation, which was not readily detected by Southern blotting and traditional PCR. It consisted of a 305 kb duplication around Poldip2 with retention of the wild type allele and could be traced back to the original targeted ES cell clone. We offer simple suggestions to rapidly detect similar accidents, which may affect genome editing using both traditional and CRISPR-based methods. In the second part of the present study, correctly targeted floxed Poldip2 mice were generated and used to produce a new constitutive knockout line by crossing with a Cre deleter. In contrast to the gene trap model, many homozygous knockout mice were viable, in spite of having no POLDIP2 expression. To further characterize the effects of Poldip2 ablation in the vasculature, RNA-seq and RT-qPCR experiments were performed in constitutive knockout arteries. Results show that POLDIP2 inactivation affects multiple cellular processes and provide new opportunities for future in-depth study of its functions., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2021
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19. Poldip2 controls leukocyte infiltration into the ischemic brain by regulating focal adhesion kinase-mediated VCAM-1 induction.
- Author
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Eidson LN, Gao Q, Qu H, Kikuchi DS, Campos ACP, Faidley EA, Sun YY, Kuan CY, Pagano RL, Lassègue B, Tansey MG, Griendling KK, and Hernandes MS
- Subjects
- Animals, Brain Ischemia genetics, Focal Adhesion Kinase 1 genetics, Ischemic Stroke genetics, Mice, Mice, Mutant Strains, Mitochondrial Proteins genetics, Nuclear Proteins genetics, Vascular Cell Adhesion Molecule-1 genetics, Brain metabolism, Brain Ischemia metabolism, Focal Adhesion Kinase 1 metabolism, Ischemic Stroke metabolism, Leukocytes metabolism, Mitochondrial Proteins metabolism, Nuclear Proteins metabolism, Vascular Cell Adhesion Molecule-1 metabolism
- Abstract
Stroke is a multiphasic process involving a direct ischemic brain injury which is then exacerbated by the influx of immune cells into the brain tissue. Activation of brain endothelial cells leads to the expression of adhesion molecules such vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells, further increasing leukocyte recruitment. Polymerase δ-interacting protein 2 (Poldip2) promotes brain vascular inflammation and leukocyte recruitment via unknown mechanisms. This study aimed to define the role of Poldip2 in mediating vascular inflammation and leukocyte recruitment following cerebral ischemia. Cerebral ischemia was induced in Poldip2
+/+ and Poldip2+/- mice and brains were isolated and processed for flow cytometry or RT-PCR. Cultured rat brain microvascular endothelial cells were used to investigate the effect of Poldip2 depletion on focal adhesion kinase (FAK)-mediated VCAM-1 induction. Poldip2 depletion in vivo attenuated the infiltration of myeloid cells, inflammatory monocytes/macrophages and decreased the induction of adhesion molecules. Focusing on VCAM-1, we demonstrated mechanistically that FAK activation was a critical intermediary in Poldip2-mediated VCAM-1 induction. In conclusion, Poldip2 is an important mediator of endothelial dysfunction and leukocyte recruitment. Thus, Poldip2 could be a therapeutic target to improve morbidity following ischemic stroke.- Published
- 2021
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20. β 1- and β 2-integrins: central players in regulating vascular permeability and leukocyte recruitment during acute inflammation.
- Author
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Ou Z, Dolmatova E, Lassègue B, and Griendling KK
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- Animals, Cell Adhesion, Cell Communication, Endothelium, Vascular immunology, Endothelium, Vascular physiopathology, Humans, Inflammation immunology, Inflammation physiopathology, Leukocyte Rolling, Leukocytes immunology, Signal Transduction, Transendothelial and Transepithelial Migration, CD18 Antigens metabolism, Capillary Permeability, Chemotaxis, Leukocyte, Endothelium, Vascular metabolism, Inflammation metabolism, Inflammation Mediators metabolism, Integrin beta1 metabolism, Leukocytes metabolism
- Abstract
The integrin family, an indispensable part of cell-cell and cell-matrix interactions, consists of a group of heterodimeric adhesion receptors formed by α- and β-integrin subunits. Their wide expression and unique bidirectional signaling pathways allow them to play roles in a variety of biological activities including blood clot formation, cell attachment, and migration. Evidence suggests that integrins are essential regulators of the initiation of acute inflammation, especially two key aspects of this process i.e., vascular permeability and leukocyte recruitment. This mini-review discusses the importance of integrins at the onset of the acute inflammatory response and outlines research advances regarding the function of integrins and their modulators at different stages of this process. Insights into the fine-tuning of integrin signaling during acute inflammation may inspire the design of new drugs for inflammatory diseases.
- Published
- 2021
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21. Correction: Platelet-derived growth factor (PDGF) regulates slingshot phosphatase activity via Nox1-dependent auto-dephosphorylation of serine 834 in vascular smooth muscle cells.
- Author
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Maheswaranathan M, Gole HKA, Fernandez I, Lassègue B, Griendling KK, and San Martín A
- Published
- 2020
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22. Poldip2 mediates blood-brain barrier disruption in a model of sepsis-associated encephalopathy.
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Kikuchi DS, Campos ACP, Qu H, Forrester SJ, Pagano RL, Lassègue B, Sadikot RT, Griendling KK, and Hernandes MS
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- Animals, Blood-Brain Barrier drug effects, Blood-Brain Barrier pathology, Capillary Permeability drug effects, Capillary Permeability physiology, Cyclooxygenase 2 metabolism, Dinoprostone metabolism, Disease Models, Animal, Endothelial Cells drug effects, Endothelial Cells metabolism, Endothelial Cells pathology, Female, Lipopolysaccharides pharmacology, Male, Mice, Mice, Knockout, Mitochondrial Proteins genetics, NF-kappa B metabolism, Nuclear Proteins genetics, Permeability, Sepsis-Associated Encephalopathy genetics, Sepsis-Associated Encephalopathy pathology, Blood-Brain Barrier metabolism, Mitochondrial Proteins metabolism, Nuclear Proteins metabolism, Sepsis-Associated Encephalopathy metabolism
- Abstract
Background: Sepsis-associated encephalopathy (SAE), a diffuse cerebral dysfunction in the absence of direct CNS infection, is associated with increased rates of mortality and morbidity in patients with sepsis. Increased cytokine production and disruption of the blood-brain barrier (BBB) are implicated in the pathogenesis of SAE. The induction of pro-inflammatory mediators is driven, in part, by activation of NF-κΒ. Lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, potently activates NF-κΒ and its downstream targets, including cyclooxygenase-2 (Cox-2). Cox-2 catalyzes prostaglandin synthesis and in the brain prostaglandin, E2 is capable of inducing endothelial permeability. Depletion of polymerase δ-interacting protein 2 (Poldip2) has previously been reported to attenuate BBB disruption, possibly via regulation of NF-κΒ, in response to ischemic stroke. Here we investigated Poldip2 as a novel regulator of NF-κΒ/cyclooxygenase-2 signaling in an LPS model of SAE., Methods: Intraperitoneal injections of LPS (18 mg/kg) were used to induce BBB disruption in Poldip2
+/+ and Poldip2+/- mice. Changes in cerebral vascular permeability and the effect of meloxicam, a selective Cox-2 inhibitor, were assessed by Evans blue dye extravasation. Cerebral cortices of Poldip2+/+ and Poldip2+/- mice were further evaluated by immunoblotting and ELISA. To investigate the role of endothelial Poldip2, immunofluorescence microscopy and immunoblotting were performed to study the effect of siPoldip2 on LPS-mediated NF-κΒ subunit p65 translocation and Cox-2 induction in rat brain microvascular endothelial cells. Finally, FITC-dextran transwell assay was used to assess the effect of siPoldip2 on LPS-induced endothelial permeability., Results: Heterozygous deletion of Poldip2 conferred protection against LPS-induced BBB permeability. Alterations in Poldip2+/+ BBB integrity were preceded by induction of Poldip2, p65, and Cox-2, which was not observed in Poldip2+/- mice. Consistent with these findings, prostaglandin E2 levels were significantly elevated in Poldip2+/+ cerebral cortices compared to Poldip2+/- cortices. Treatment with meloxicam attenuated LPS-induced BBB permeability in Poldip2+/+ mice, while having no significant effect in Poldip2+/- mice. Moreover, silencing of Poldip2 in vitro blocked LPS-induced p65 nuclear translocation, Cox-2 expression, and endothelial permeability., Conclusions: These data suggest Poldip2 mediates LPS-induced BBB disruption by regulating NF-κΒ subunit p65 activation and Cox-2 and prostaglandin E2 induction. Consequently, targeted inhibition of Poldip2 may provide clinical benefit in the prevention of sepsis-induced BBB disruption.- Published
- 2019
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23. Polymerase-δ-interacting protein 2 activates the RhoGEF epithelial cell transforming sequence 2 in vascular smooth muscle cells.
- Author
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Huff LP, Kikuchi DS, Faidley E, Forrester SJ, Tsai MZ, Lassègue B, and Griendling KK
- Subjects
- Animals, Cell Proliferation physiology, HEK293 Cells, Humans, Male, Mice, Mice, Inbred C57BL, Muscle, Smooth, Vascular cytology, Nuclear Proteins antagonists & inhibitors, Proto-Oncogene Proteins antagonists & inhibitors, RNA, Small Interfering pharmacology, Rats, Rats, Sprague-Dawley, Muscle, Smooth, Vascular metabolism, Nuclear Proteins metabolism, Proto-Oncogene Proteins metabolism, Rho Guanine Nucleotide Exchange Factors metabolism
- Abstract
Polymerase-δ-interacting protein 2 (Poldip2) controls a wide variety of cellular functions and vascular pathologies. To mediate these effects, Poldip2 interacts with numerous proteins and generates reactive oxygen species via the enzyme NADPH oxidase 4 (Nox4). We have previously shown that Poldip2 can activate the Rho family GTPase RhoA, another signaling node within the cell. In this study, we aimed to better understand how Poldip2 activates Rho family GTPases and the functions of the involved proteins in vascular smooth muscle cells (VSMCs). RhoA is activated by guanine nucleotide exchange factors. Using nucleotide-free RhoA (isolated from bacteria) to pulldown active RhoGEFs, we found that the RhoGEF epithelial cell transforming sequence 2 (Ect2) is activated by Poldip2. Ect2 is a critical RhoGEF for Poldip2-mediated RhoA activation, because siRNA against Ect2 prevented Poldip2-mediated RhoA activity (measured by rhotekin pulldowns). Surprisingly, we were unable to detect a direct interaction between Poldip2 and Ect2, as they did not coimmunoprecipitate. Nox4 is not required for Poldip2-driven Ect2 activation, as Poldip2 overexpression induced Ect2 activation in Nox4 knockout VSMCs similar to wild-type cells. However, antioxidant treatment blocked Poldip2-induced Ect2 activation. This indicates a novel reactive oxygen species-driven mechanism by which Poldip2 regulates Rho family GTPases. Finally, we examined the function of these proteins in VSMCs, using siRNA against Poldip2 or Ect2 and determined that Poldip2 and Ect2 are both essential for vascular smooth muscle cell cytokinesis and proliferation.
- Published
- 2019
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24. Poldip2 knockdown inhibits vascular smooth muscle proliferation and neointima formation by regulating the expression of PCNA and p21.
- Author
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Datla SR, L Hilenski L, Seidel-Rogol B, Dikalova AE, Harousseau M, Punkova L, Joseph G, Taylor WR, Lassègue B, and Griendling KK
- Subjects
- Animals, Atherosclerosis genetics, Atherosclerosis metabolism, Atherosclerosis pathology, Carrier Proteins genetics, Carrier Proteins metabolism, Cell Proliferation genetics, Down-Regulation, Gene Knockdown Techniques, Humans, Mice, Mice, Knockout, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Myocytes, Smooth Muscle metabolism, Myocytes, Smooth Muscle pathology, Neointima pathology, Neointima prevention & control, Nuclear Proteins genetics, Nuclear Proteins metabolism, RNA, Small Interfering genetics, Rats, Superoxides metabolism, Carrier Proteins antagonists & inhibitors, Cell Proliferation physiology, Cyclin-Dependent Kinase Inhibitor p21 metabolism, Mitochondrial Proteins deficiency, Muscle, Smooth, Vascular metabolism, Muscle, Smooth, Vascular pathology, Neointima metabolism, Nuclear Proteins antagonists & inhibitors, Nuclear Proteins deficiency, Proliferating Cell Nuclear Antigen metabolism
- Abstract
Polymerase delta-interacting protein 2 (Poldip2) is a multi-functional protein with numerous roles in the vasculature, including the regulation of cell apoptosis and migration, as well as extracellular matrix deposition; however, its role in VSMC proliferation and neointimal formation is unknown. In this study, we investigated the role of Poldip2 in intraluminal wire-injury induced neointima formation and proliferation of vascular smooth muscle cells in vitro and in vivo. Poldip2 expression was observed in the intima and media of human atherosclerotic arteries, where it colocalized with proliferating cell nuclear antigen (PCNA). Wire injury of femoral arteries of Poldip2
+/+ mice induced robust neointimal formation after 2 weeks, which was impaired in Poldip2+/‒ mice. PCNA expression was significantly reduced and expression of the cell cycle inhibitor p21 was significantly increased in wire-injured arteries of Poldip2+/‒ animals compared to wild-type controls. No difference was observed in apoptosis. Downregulation of Poldip2 in rat aortic smooth muscle cells significantly reduced serum-induced proliferation and PCNA expression, but upregulated p21 expression. Downregulation of p21 using siRNA reversed the inhibition of proliferation induced by knockdown of Poldip2. These results indicate that Poldip2 plays a critical role in the proliferation of VSMCs.- Published
- 2019
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25. The cofilin phosphatase slingshot homolog 1 restrains angiotensin II-induced vascular hypertrophy and fibrosis in vivo.
- Author
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Williams HC, Ma J, Weiss D, Lassègue B, Sutliff RL, and San Martín A
- Subjects
- Animals, Aorta metabolism, Aorta pathology, Disease Models, Animal, Female, Fibrosis, Hypertension etiology, Hypertension metabolism, Hypertension pathology, Hypertrophy, Male, Mice, Mice, 129 Strain, Mice, Inbred C57BL, Mice, Knockout, Phosphoprotein Phosphatases deficiency, Phosphoprotein Phosphatases genetics, Transforming Growth Factor beta1 metabolism, Vascular Remodeling genetics, Angiotensin II metabolism, Phosphoprotein Phosphatases metabolism, Vascular Remodeling physiology
- Abstract
The dual specificity phosphatase slingshot homolog 1 (SSH1) contributes to actin remodeling by dephosphorylating and activating the actin-severing protein cofilin. The reorganization of the actin cytoskeleton has been implicated in chronic hypertension and the subsequent mechano-adaptive rearrangement of vessel wall components. Therefore, using a novel Ssh1
-/- mouse model, we investigated the potential role of SSH1 in angiotensin II (Ang II)-induced hypertension, and vascular remodeling. We found that loss of SSH1 did not produce overt phenotypic changes and that baseline blood pressures as well as heart rates were comparable between Ssh1+/+ and Ssh1-/- mice. Although 14 days of Ang II treatment equally increased systolic blood pressure in both genotypes, histological assessment of aortic samples indicated that medial thickening was exacerbated by the loss of SSH1. Consequently, reverse-transcription quantitative PCR analysis of the transcripts from Ang II-infused animals confirmed increased aortic expression levels of fibronectin, and osteopontin in Ssh1-/- when compared to wild-type mice. Mechanistically, our data suggest that fibrosis in SSH1-deficient mice occurs by a process that involves aberrant responses to Ang II-induced TGFβ1. Taken together, our work indicates that Ang II-dependent fibrotic gene expression and vascular remodeling, but not the Ang II-induced pressor response, are modulated by SSH1-mediated signaling pathways and SSH1 activity is protective against Ang II-induced remodeling in the vasculature.- Published
- 2019
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26. Poldip2 deficiency protects against lung edema and vascular inflammation in a model of acute respiratory distress syndrome.
- Author
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Forrester SJ, Xu Q, Kikuchi DS, Okwan-Duodu D, Campos AC, Faidley EA, Zhang G, Lassègue B, Sadikot RT, Griendling KK, and Hernandes MS
- Subjects
- Animals, Cell Adhesion, Coculture Techniques, Cytokines metabolism, Disease Models, Animal, Endothelial Cells pathology, Female, Humans, Leukocytes metabolism, Leukocytes pathology, Male, Mice, Inbred C57BL, Mitochondrial Proteins genetics, Nuclear Proteins genetics, Nuclear Proteins metabolism, Pulmonary Edema genetics, Pulmonary Edema metabolism, Pulmonary Edema pathology, Reactive Oxygen Species metabolism, Respiratory Distress Syndrome genetics, Respiratory Distress Syndrome pathology, Signal Transduction, THP-1 Cells, Vascular Cell Adhesion Molecule-1 metabolism, Vasculitis genetics, Vasculitis metabolism, Vasculitis pathology, Capillary Permeability, Endothelial Cells metabolism, Lung blood supply, Mitochondrial Proteins deficiency, Nuclear Proteins deficiency, Pulmonary Edema prevention & control, Respiratory Distress Syndrome metabolism, Vasculitis prevention & control
- Abstract
Acute respiratory distress syndrome (ARDS) in a deadly disease that can be brought on by endotoxins such as lipopolysaccharide (LPS). ARDS is characterized by vascular permeability, a severe inflammatory response, lung leukocyte infiltration, and resultant lung edema. Polymerase δ-interacting protein 2 (Poldip2) is a novel regulator of blood-brain barrier permeability; however, its role in regulating lung permeability and vascular inflammation is unknown. Here, the role of Poldip2 in regulating vascular permeability and inflammation in a mouse model of ARDS was assessed. Heterozygous deletion of Poldip2 was found to reduce LPS-induced mortality within 20 h, lung inflammatory signaling, and leukocyte infiltration. Moreover, reduced Poldip2-suppressed LP-induced vascular cell adhesion molecule (VCAM)-1 induction, leukocyte recruitment, and mitochondrial reactive oxygen species (ROS) production in vitro These data indicate that Poldip2 is an important regulator of the debilitating consequences of ARDS, potentially through the regulation of mitochondrial ROS-induced inflammatory signaling. Consequently, inhibition of Poldip2 may be a viable option for therapeutic discovery moving forward., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)
- Published
- 2019
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27. NOX4 (NADPH Oxidase 4) and Poldip2 (Polymerase δ-Interacting Protein 2) Induce Filamentous Actin Oxidation and Promote Its Interaction With Vinculin During Integrin-Mediated Cell Adhesion.
- Author
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Vukelic S, Xu Q, Seidel-Rogol B, Faidley EA, Dikalova AE, Hilenski LL, Jorde U, Poole LB, Lassègue B, Zhang G, and Griendling KK
- Subjects
- Actin Cytoskeleton genetics, Animals, Carrier Proteins genetics, Cell Movement, Cells, Cultured, Humans, Hydrogen Peroxide metabolism, Muscle, Smooth, Vascular ultrastructure, Myocytes, Smooth Muscle ultrastructure, NADPH Oxidase 4 genetics, Nuclear Proteins genetics, Oxidation-Reduction, Rats, Signal Transduction, Actin Cytoskeleton enzymology, Carrier Proteins metabolism, Cell Adhesion, Integrins metabolism, Muscle, Smooth, Vascular enzymology, Myocytes, Smooth Muscle enzymology, NADPH Oxidase 4 metabolism, Nuclear Proteins metabolism, Vinculin metabolism
- Abstract
Objective- Actin cytoskeleton assembly and organization, as a result of focal adhesion (FA) formation during cell adhesion, are dependent on reactive oxygen species and the cellular redox environment. Poldip2 (polymerase δ-interacting protein 2), a novel regulator of NOX4 (NADPH oxidase 4), plays a significant role in reactive oxygen species production and cytoskeletal remodeling. Thus, we hypothesized that endogenous reactive oxygen species derived from Poldip2/NOX4 contribute to redox regulation of actin and cytoskeleton assembly during integrin-mediated cell adhesion. Approach and Results- Using vascular smooth muscle cells, we verified that hydrogen peroxide (H
2 O2 ) levels increase during integrin-mediated cell attachment as a result of activation of NOX4. Filamentous actin (F-actin) was oxidized by sulfenylation during cell attachment, with a peak at 3 hours (0.80±0.04 versus 0.08±0.13 arbitrary units at time zero), which was enhanced by overexpression of Poldip2. Depletion of Poldip2 or NOX4 using siRNA, or scavenging of endogenous H2 O2 with catalase, inhibited F-actin oxidation by 78±26%, 99±1%, and 98±1%, respectively. To determine the consequence of F-actin oxidation, we examined the binding of F-actin to vinculin, a protein involved in FA complexes that regulates FA maturation. Vinculin binding during cell adhesion as well as migration capacity were inhibited after transfection with actin containing 2 oxidation-resistant point mutations (C272A and C374A). Silencing of Poldip2 or NOX4 also impaired actin-vinculin interaction, which disturbed maturation of FAs and inhibited cell migration. Conclusions- These results suggest that integrin engagement during cell attachment activates Poldip2/Nox4 to oxidize actin, which modulates FA assembly.- Published
- 2018
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28. Design, synthesis, and biological evaluation of inhibitors of the NADPH oxidase, Nox4.
- Author
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Xu Q, Kulkarni AA, Sajith AM, Hussein D, Brown D, Güner OF, Reddy MD, Watkins EB, Lassègue B, Griendling KK, and Bowen JP
- Subjects
- Cell Line, Cell Survival drug effects, Enzyme Inhibitors chemistry, Enzyme Inhibitors pharmacology, Humans, NADPH Oxidase 4 metabolism, Reactive Oxygen Species metabolism, Signal Transduction drug effects, Sulfonylurea Compounds chemical synthesis, Sulfonylurea Compounds pharmacology, Drug Design, Enzyme Inhibitors chemical synthesis, NADPH Oxidase 4 antagonists & inhibitors, Sulfonylurea Compounds chemistry
- Abstract
NADPH oxidases (Nox enzymes) are critical mediators of both physiologic and pathophysiologic processes. Nox enzymes catalyze NADPH-dependent generation of reactive oxygen species (ROS), including superoxide and hydrogen peroxide. Until recently, Nox4 was proposed to be involved exclusively in normal physiologic functions. Compelling evidence, however, suggests that Nox4 plays a critical role in fibrosis, as well as a host of pathologies and diseases. These considerations led to a search for novel, small molecule inhibitors of this important enzyme. Ultimately, a series of novel tertiary sulfonylureas (23-25) was designed using pharmacophore modeling, synthesized, and evaluated for inhibition of Nox4-dependent signaling., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2018
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29. Poldip2 is an oxygen-sensitive protein that controls PDH and αKGDH lipoylation and activation to support metabolic adaptation in hypoxia and cancer.
- Author
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Paredes F, Sheldon K, Lassègue B, Williams HC, Faidley EA, Benavides GA, Torres G, Sanhueza-Olivares F, Yeligar SM, Griendling KK, Darley-Usmar V, and San Martin A
- Subjects
- Animals, Coenzyme A Ligases genetics, Coenzyme A Ligases metabolism, Humans, Hypoxia genetics, Hypoxia metabolism, Ketoglutarate Dehydrogenase Complex genetics, Ketoglutarate Dehydrogenase Complex metabolism, Lipoylation, Mice, Mice, Inbred C57BL, Mice, Knockout, Mitochondria enzymology, Mitochondria genetics, Mitochondria metabolism, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Neoplasms genetics, Neoplasms metabolism, Nuclear Proteins genetics, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases metabolism, Pyruvate Dehydrogenase Acetyl-Transferring Kinase, Thioctic Acid metabolism, Hypoxia enzymology, Neoplasms enzymology, Nuclear Proteins metabolism, Oxygen metabolism
- Abstract
Although the addition of the prosthetic group lipoate is essential to the activity of critical mitochondrial catabolic enzymes, its regulation is unknown. Here, we show that lipoylation of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (αKDH) complexes is a dynamically regulated process that is inhibited under hypoxia and in cancer cells to restrain mitochondrial respiration. Mechanistically, we found that the polymerase-δ interacting protein 2 (Poldip2), a nuclear-encoded mitochondrial protein of unknown function, controls the lipoylation of the pyruvate and α-KDH dihydrolipoamide acetyltransferase subunits by a mechanism that involves regulation of the caseinolytic peptidase (Clp)-protease complex and degradation of the lipoate-activating enzyme Ac-CoA synthetase medium-chain family member 1 (ACSM1). ACSM1 is required for the utilization of lipoic acid derived from a salvage pathway, an unacknowledged lipoylation mechanism. In Poldip2-deficient cells, reduced lipoylation represses mitochondrial function and induces the stabilization of hypoxia-inducible factor 1α (HIF-1α) by loss of substrate inhibition of prolyl-4-hydroxylases (PHDs). HIF-1α-mediated retrograde signaling results in a metabolic reprogramming that resembles hypoxic and cancer cell adaptation. Indeed, we observe that Poldip2 expression is down-regulated by hypoxia in a variety of cell types and basally repressed in triple-negative cancer cells, leading to inhibition of lipoylation of the pyruvate and α-KDH complexes and mitochondrial dysfunction. Increasing mitochondrial lipoylation by forced expression of Poldip2 increases respiration and reduces the growth rate of cancer cells. Our work unveils a regulatory mechanism of catabolic enzymes required for metabolic plasticity and highlights the role of Poldip2 as key during hypoxia and cancer cell metabolic adaptation., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)
- Published
- 2018
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30. Polymerase delta-interacting protein 2 deficiency protects against blood-brain barrier permeability in the ischemic brain.
- Author
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Hernandes MS, Lassègue B, Hilenski LL, Adams J, Gao N, Kuan CY, Sun YY, Cheng L, Kikuchi DS, Yepes M, and Griendling KK
- Subjects
- Animals, Brain diagnostic imaging, Brain metabolism, Brain Ischemia diagnostic imaging, Cells, Cultured, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Blood-Brain Barrier metabolism, Brain Ischemia metabolism, Brain Ischemia prevention & control, Capillary Permeability physiology, Mitochondrial Proteins deficiency, Neuroprotection physiology, Nuclear Proteins deficiency
- Abstract
Background: Polymerase δ-interacting protein 2 (Poldip2) is a multifunctional protein that regulates vascular extracellular matrix composition and matrix metalloproteinase (MMP) activity. The blood-brain barrier (BBB) is a dynamic system assembled by endothelial cells, basal lamina, and perivascular astrocytes, raising the possibility that Poldip2 may be involved in maintaining its structure. We investigated the role of Poldip2 in the late BBB permeability induced by cerebral ischemia., Methods: Transient middle cerebral artery occlusion (tMCAO) was induced in Poldip2
+/+ and Poldip2+/- mice. The volume of the ischemic lesion was measured in triphenyltetrazolium chloride-stained sections. BBB breakdown was evaluated by Evans blue dye extravasation. Poldip2 protein expression was evaluated by western blotting. RT-PCR, zymography, and ELISAs were used to measure mRNA levels, activity, and protein levels of cytokines and MMPs. Cultured astrocytes were transfected with Poldip2 siRNA, and mRNA levels of cytokines were evaluated as well as IκBα protein degradation., Results: Cerebral ischemia induced the expression of Poldip2. Compared to Poldip2+/+ mice, Poldip2+/- animals exhibited decreased Evans blue dye extravasation and improved survival 24 h following stroke. Poldip2 expression was upregulated in astrocytes exposed to oxygen and glucose deprivation (OGD) and siRNA-mediated downregulation of Poldip2 abrogated OGD-induced IL-6 and TNF-α expression. In addition, siRNA against Poldip2 inhibited TNF-α-induced IκBα degradation. TNF-α, IL-6, MCP-1, VEGF, and MMP expression induced by cerebral ischemia was abrogated in Poldip2+/- mice. The protective effect of Poldip2 depletion on the increased permeability of the BBB was partially reversed by systemic administration of TNF-α., Conclusions: Poldip2 is upregulated following ischemic stroke and mediates the breakdown of the BBB by increasing cerebral cytokine production and MMP activation. Therefore, Poldip2 appears to be a promising novel target for the development of therapeutic strategies to prevent the development of cerebral edema in the ischemic brain.- Published
- 2018
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31. Polymerase δ-interacting Protein 2: A Multifunctional Protein.
- Author
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Hernandes MS, Lassègue B, and Griendling KK
- Subjects
- Animals, Cardiovascular Diseases metabolism, Cardiovascular Diseases physiopathology, Gene Expression Regulation, Humans, Kidney Diseases metabolism, Kidney Diseases physiopathology, Models, Molecular, Neurodegenerative Diseases metabolism, Neurodegenerative Diseases physiopathology, Nuclear Proteins chemistry, Nuclear Proteins genetics, Protein Conformation, Structure-Activity Relationship, Nuclear Proteins metabolism, Signal Transduction
- Abstract
Polymerase δ-interacting protein 2 (Poldip2) is a multifunctional protein originally described as a binding partner of the p50 subunit of DNA polymerase δ and proliferating cell nuclear antigen. In addition to its role in DNA replication and damage repair, Poldip2 has been implicated in mitochondrial function, extracellular matrix regulation, cell cycle progression, focal adhesion turnover, and cell migration. However, Poldip2 functions are incompletely understood. In this review, we discuss recent literature on Poldip2 tissue distribution, subcellular localization, and function. We also address the putative function of Poldip2 in cardiovascular disease, neurodegenerative conditions and in renal pathophysiology.
- Published
- 2017
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32. Cyclic Strain and Hypertension Increase Osteopontin Expression in the Aorta.
- Author
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Caesar C, Lyle AN, Joseph G, Weiss D, Alameddine FMF, Lassègue B, Griendling KK, and Taylor WR
- Abstract
Hypertension has a direct impact on vascular hypertrophy and is a known risk factor for the development of atherosclerosis. Osteopontin (OPN) has emerged as an important protein mediator of inflammation and remodeling of large arteries. However, its role and mechanism of regulation in the setting of hypertension is still unknown. Our objectives for this study were therefore to investigate the role of OPN in hypertension-induced vascular remodeling and inflammation. OPN Knockout (KO) and wild type (WT) mice were made hypertensive with angiotensin II (Ang II) infusion for seven days. We observed that OPN KO aortas were protected against Ang II-induced medial hypertrophy and inflammation, despite comparable increases in systolic blood pressure (SBP) in both groups. OPN expression was increased in WT aortas from hypertensive mice (induced by either Ang II or norepinephrine). OPN expression was increased in aortic smooth muscle cells (SMCs) subjected to cyclic mechanical strain suggesting that mechanical deformation of the aortic wall is responsible in part for the increased OPN expression induced by hypertension. Finally, we utilized hypertensive transgenic smooth muscle cell-specific catalase overexpressing (Tg
SMC-Cat ) mice to determine the role of H2 O2 in mediating hypertension-induced increases in OPN expression. We also found that the hypertension-induced increase in OPN expression was inhibited in transgenic smooth muscle cell-specific catalase overexpressing (TgSMC-Cat ) mice, suggesting that H2 O2 , plays a vital role in mediating the hypertension-induced increase in OPN expression. Taken together, these results define a potentially important role for OPN in the pathophysiology of hypertension., Competing Interests: CONFLICT OF INTEREST Christa Caesar, Alicia N. Lyle, Giji Joseph, Daiana Weiss, Fadi M. F. Alameddine, Bernard Lassègue, Kathy K. Griendling, and W. Robert Taylor have no conflicts of interest to disclose.- Published
- 2017
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33. NOX4-derived reactive oxygen species limit fibrosis and inhibit proliferation of vascular smooth muscle cells in diabetic atherosclerosis.
- Author
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Di Marco E, Gray SP, Kennedy K, Szyndralewiez C, Lyle AN, Lassègue B, Griendling KK, Cooper ME, Schmidt HHHW, and Jandeleit-Dahm KAM
- Subjects
- Animals, Aorta metabolism, Aorta pathology, Atherosclerosis etiology, Atherosclerosis pathology, Becaplermin, Cell Proliferation, Cells, Cultured, Diabetes Mellitus, Experimental complications, Diabetes Mellitus, Experimental pathology, Fibrosis, Male, Mice, 129 Strain, Mice, Inbred C57BL, Mice, Knockout, Muscle, Smooth, Vascular metabolism, Muscle, Smooth, Vascular pathology, NADPH Oxidase 1 metabolism, NADPH Oxidase 4 genetics, Osteopontin genetics, Osteopontin metabolism, Proto-Oncogene Proteins c-sis genetics, Proto-Oncogene Proteins c-sis metabolism, Superoxides metabolism, Atherosclerosis enzymology, Diabetes Mellitus, Experimental enzymology, Myocytes, Smooth Muscle physiology, NADPH Oxidase 4 metabolism, Reactive Oxygen Species metabolism
- Abstract
Smooth muscle cell (SMC) proliferation and fibrosis contribute to the development of advanced atherosclerotic lesions. Oxidative stress caused by increased production or unphysiological location of reactive oxygen species (ROS) is a known major pathomechanism. However, in atherosclerosis, in particular under hyperglycaemic/diabetic conditions, the hydrogen peroxide-producing NADPH oxidase type 4 (NOX4) is protective. Here we aim to elucidate the mechanisms underlying this paradoxical atheroprotection of vascular smooth muscle NOX4 under conditions of normo- and hyperglycaemia both in vivo and ex vivo. Following 20-weeks of streptozotocin-induced diabetes, Apoe(-/-) mice showed a reduction in SM-alpha-actin and calponin gene expression with concomitant increases in platelet-derived growth factor (PDGF), osteopontin (OPN) and the extracellular matrix (ECM) protein fibronectin when compared to non-diabetic controls. Genetic deletion of Nox4 (Nox4(-/)(-)Apoe(-/-)) exacerbated diabetes-induced expression of PDGF, OPN, collagen I, and proliferation marker Ki67. Aortic SMCs isolated from NOX4-deficient mice exhibited a dedifferentiated phenotype including loss of contractile gene expression, increased proliferation and ECM production as well as elevated levels of NOX1-associated ROS. Mechanistic studies revealed that elevated PDGF signalling in NOX4-deficient SMCs mediated the loss of calponin and increase in fibronectin, while the upregulation of NOX1 was associated with the increased expression of OPN and markers of proliferation. These findings demonstrate that NOX4 actively regulates SMC pathophysiological responses in diabetic Apoe(-/-) mice and in primary mouse SMCs through the activities of PDGF and NOX1., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
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34. Polymerase delta-interacting protein 2 regulates collagen accumulation via activation of the Akt/mTOR pathway in vascular smooth muscle cells.
- Author
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Fujii M, Amanso A, Abrahão TB, Lassègue B, and Griendling KK
- Subjects
- Animals, Aorta growth & development, Aorta metabolism, Cell Proliferation genetics, Fibronectins metabolism, Integrin beta Chains metabolism, Mice, Mitochondrial Proteins metabolism, Myocytes, Smooth Muscle metabolism, Nuclear Proteins metabolism, Phosphatidylinositol 3-Kinases biosynthesis, Proteasome Endopeptidase Complex metabolism, Protein Biosynthesis genetics, Proteolysis, Proto-Oncogene Proteins c-akt metabolism, Signal Transduction, TOR Serine-Threonine Kinases genetics, Collagen Type I metabolism, Extracellular Matrix genetics, Integrin beta Chains biosynthesis, Mitochondrial Proteins genetics, Muscle, Smooth, Vascular metabolism, Nuclear Proteins genetics
- Abstract
Objectives: Polymerase delta interacting protein 2 (Poldip2) has previously been implicated in migration, proliferation and extracellular matrix (ECM) production in vascular smooth muscle cells. To better understand the role of Poldip2 in ECM regulation, we investigated the mechanism responsible for collagen I accumulation in Poldip2(+/-) mouse aortic smooth muscle cells (MASMs)., Approach and Results: Protein degradation and protein synthesis pathways were investigated. Depletion of Poldip2 had no effect on proteasome activity, but caused a partial reduction in autophagic flux. However, the rate of collagen I degradation was increased in Poldip2(+/-) vs. Poldip2(+/+) MASMs. Conversely, activation of the PI3K/Akt/mTOR signaling pathway, involved in regulation of protein synthesis, was significantly elevated in Poldip2(+/-) MASMs as was β1-integrin expression. Suppressing mTOR signaling using Akt inhibitor or rapamycin and reducing β1-integrin expression using siRNA prevented the increase in collagen I production. While collagen I and fibronectin were increased in Poldip2(+/-) MASMs, overall protein synthesis was not different from that in Poldip2(+/)(+)MASMs, suggesting selectivity of Poldip2 for ECM proteins., Conclusions: Poldip2(+/-) MASMs exhibit higher β1-integrin expression and activity of the PI3K/Akt/mTOR signaling pathway, leading to increased ECM protein synthesis. These findings have important implications for vascular diseases in which ECM accumulation plays a role., (Copyright © 2016 Elsevier Ltd. All rights reserved.)
- Published
- 2016
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35. Hic-5 Mediates TGFβ-Induced Adhesion in Vascular Smooth Muscle Cells by a Nox4-Dependent Mechanism.
- Author
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Fernandez I, Martin-Garrido A, Zhou DW, Clempus RE, Seidel-Rogol B, Valdivia A, Lassègue B, García AJ, Griendling KK, and San Martin A
- Subjects
- Cell Adhesion genetics, Cell Adhesion physiology, Cell Movement genetics, Cell Movement physiology, Cells, Cultured, Focal Adhesions genetics, Focal Adhesions physiology, HSP27 Heat-Shock Proteins metabolism, Humans, Intracellular Signaling Peptides and Proteins genetics, LIM Domain Proteins genetics, Muscle, Smooth, Vascular cytology, NADPH Oxidase 4, NADPH Oxidases genetics, Sensitivity and Specificity, Signal Transduction, HSP27 Heat-Shock Proteins genetics, Intracellular Signaling Peptides and Proteins metabolism, LIM Domain Proteins metabolism, NADPH Oxidases metabolism, Transforming Growth Factor beta metabolism
- Abstract
Objective: Focal adhesions (FAs) link the cytoskeleton to the extracellular matrix and as such play important roles in growth, migration, and contractile properties of vascular smooth muscle cells. Recently, it has been shown that downregulation of Nox4, a transforming growth factor (TGF) β-inducible, hydrogen peroxide (H2O2)-producing enzyme, affects the number of FAs. However, the effectors downstream of Nox4 that mediate FA regulation are unknown. The FA resident protein H2O2-inducible clone (Hic)-5 is H2O2 and TGFβ inducible, and a binding partner of the heat shock protein (Hsp) 27. The objective of this study was to elucidate the mechanism, by which Hic-5 and Hsp27 participate in TGFβ-induced, Nox4-mediated vascular smooth muscle cell adhesion and migration., Approach and Results: Through a combination of molecular biology and biochemistry techniques, we found that TGFβ, by a Nox4-dependent mechanism, induces the expression and interaction of Hic-5 and Hsp27, which is essential for Hic-5 localization to FAs. Importantly, we found that Hic-5 expression is required for the TGFβ-mediated increase in FA number, adhesive forces and migration. Mechanistically, Nox4 downregulation impedes Smad (small body size and mothers against decapentaplegic) signaling by TGFβ, and Hsp27 and Hic-5 upregulation by TGFβ is blocked in small body size and mothers against decapentaplegic 4-deficient cells., Conclusions: Hic-5 and Hsp27 are effectors of Nox4 required for TGFβ-stimulated FA formation, adhesion strength and migration in vascular smooth muscle cell., (© 2015 American Heart Association, Inc.)
- Published
- 2015
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36. Poldip2 controls vascular smooth muscle cell migration by regulating focal adhesion turnover and force polarization.
- Author
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Datla SR, McGrail DJ, Vukelic S, Huff LP, Lyle AN, Pounkova L, Lee M, Seidel-Rogol B, Khalil MK, Hilenski LL, Terada LS, Dawson MR, Lassègue B, and Griendling KK
- Subjects
- Animals, Carrier Proteins genetics, Cell Adhesion, Cell Polarity, Focal Adhesion Protein-Tyrosine Kinases metabolism, Hydrogen Peroxide metabolism, Muscle, Smooth, Vascular cytology, Muscle, Smooth, Vascular metabolism, Myocytes, Smooth Muscle drug effects, Myocytes, Smooth Muscle physiology, NADPH Oxidase 4, NADPH Oxidases genetics, NADPH Oxidases metabolism, Platelet-Derived Growth Factor pharmacology, Rats, rhoA GTP-Binding Protein metabolism, Carrier Proteins metabolism, Cell Movement, Focal Adhesions metabolism, Myocytes, Smooth Muscle metabolism
- Abstract
Polymerase-δ-interacting protein 2 (Poldip2) interacts with NADPH oxidase 4 (Nox4) and regulates migration; however, the precise underlying mechanisms are unclear. Here, we investigated the role of Poldip2 in focal adhesion turnover, as well as traction force generation and polarization. Poldip2 overexpression (AdPoldip2) in vascular smooth muscle cells (VSMCs) impairs PDGF-induced migration and induces a characteristic phenotype of long cytoplasmic extensions. AdPoldip2 also prevents the decrease in spreading and increased aspect ratio observed in response to PDGF and slightly impairs cell contraction. Moreover, AdPoldip2 blocks focal adhesion dissolution and sustains H2O2 levels in focal adhesions, whereas Poldip2 knockdown (siPoldip2) significantly decreases the number of focal adhesions. RhoA activity is unchanged when focal adhesion dissolution is stimulated in control cells but increases in AdPoldip2-treated cells. Inhibition of RhoA blocks Poldip2-mediated attenuation of focal adhesion dissolution, and overexpression of RhoA or focal adhesion kinase (FAK) reverses the loss of focal adhesions induced by siPoldip2, indicating that RhoA and FAK mediate the effect of Poldip2 on focal adhesions. Nox4 silencing prevents focal adhesion stabilization by AdPoldip2 and induces a phenotype similar to siPoldip2, suggesting a role for Nox4 in Poldip2-induced focal adhesion stability. As a consequence of impaired focal adhesion turnover, PDGF-treated AdPoldip2 cells are unable to reduce and polarize traction forces, a necessary first step in migration. These results implicate Poldip2 in VSMC migration via regulation of focal adhesion turnover and traction force generation in a Nox4/RhoA/FAK-dependent manner., (Copyright © 2014 the American Physiological Society.)
- Published
- 2014
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37. Polymerase δ-interacting protein 2 promotes postischemic neovascularization of the mouse hindlimb.
- Author
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Amanso AM, Lassègue B, Joseph G, Landázuri N, Long JS, Weiss D, Taylor WR, and Griendling KK
- Subjects
- Animals, Apoptosis, Cell Proliferation, Cells, Cultured, Collateral Circulation, Disease Models, Animal, Heterozygote, Hindlimb, Human Umbilical Vein Endothelial Cells metabolism, Human Umbilical Vein Endothelial Cells pathology, Humans, Hydrogen Peroxide metabolism, Ischemia genetics, Ischemia pathology, Ischemia physiopathology, Macrophages metabolism, Matrix Metalloproteinase 2 metabolism, Matrix Metalloproteinase 9 metabolism, Mice, Mice, Knockout, Mitochondrial Proteins deficiency, Mitochondrial Proteins genetics, Nuclear Proteins deficiency, Nuclear Proteins genetics, RNA Interference, Recovery of Function, Regional Blood Flow, Time Factors, Transfection, Ischemia metabolism, Mitochondrial Proteins metabolism, Muscle, Skeletal blood supply, Neovascularization, Physiologic, Nuclear Proteins metabolism
- Abstract
Objective: Collateral vessel formation can functionally compensate for obstructive vascular lesions in patients with atherosclerosis. Neovascularization processes are triggered by fluid shear stress, hypoxia, growth factors, chemokines, proteases, and inflammation, as well as reactive oxygen species, in response to ischemia. Polymerase δ-interacting protein 2 (Poldip2) is a multifunctional protein that regulates focal adhesion turnover and vascular smooth muscle cell migration and modifies extracellular matrix composition. We, therefore, tested the hypothesis that loss of Poldip2 impairs collateral formation., Approach and Results: The mouse hindlimb ischemia model has been used to understand mechanisms involved in postnatal blood vessel formation. Poldip2(+/-) mice were subjected to femoral artery excision, and functional and morphological analysis of blood vessel formation was performed after injury. Heterozygous deletion of Poldip2 decreased the blood flow recovery and spontaneous running activity at 21 days after injury. H2O2 production, as well as the activity of matrix metalloproteinases-2 and -9, was reduced in these animals compared with Poldip2(+/+) mice. Infiltration of macrophages in the peri-injury muscle was also decreased; however, macrophage phenotype was similar between genotypes. In addition, the formation of capillaries and arterioles was impaired, as was angiogenesis, in agreement with a decrease in proliferation observed in endothelial cells treated with small interfering RNA against Poldip2. Finally, regression of newly formed vessels and apoptosis was more pronounced in Poldip2(+/-) mice., Conclusions: Together, these results suggest that Poldip2 promotes ischemia-induced collateral vessel formation via multiple mechanisms that likely involve reactive oxygen species-dependent activation of matrix metalloproteinase activity, as well as enhanced vascular cell growth and survival., (© 2014 American Heart Association, Inc.)
- Published
- 2014
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38. Poldip2 knockout results in perinatal lethality, reduced cellular growth and increased autophagy of mouse embryonic fibroblasts.
- Author
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Brown DI, Lassègue B, Lee M, Zafari R, Long JS, Saavedra HI, and Griendling KK
- Subjects
- Animals, Apoptosis, Cell Cycle, Cell Proliferation, DNA Repair, DNA Replication, Female, Gene Silencing, Genotype, Homozygote, Mice, Mice, Inbred C57BL, Mice, Knockout, Phosphorylation, Proliferating Cell Nuclear Antigen metabolism, Signal Transduction, Autophagy, Fibroblasts cytology, Gene Expression Regulation, Developmental, Mitochondrial Proteins metabolism, Nuclear Proteins metabolism
- Abstract
Polymerase-δ interacting protein 2 (Poldip2) is an understudied protein, originally described as a binding partner of polymerase delta and proliferating cell nuclear antigen (PCNA). Numerous roles for Poldip2 have been proposed, including mitochondrial elongation, DNA replication/repair and ROS production via Nox4. In this study, we have identified a novel role for Poldip2 in regulating the cell cycle. We used a Poldip2 gene-trap mouse and found that homozygous animals die around the time of birth. Poldip2-/- embryos are significantly smaller than wild type or heterozygous embryos. We found that Poldip2-/- mouse embryonic fibroblasts (MEFs) exhibit reduced growth as measured by population doubling and growth curves. This effect is not due to apoptosis or senescence; however, Poldip2-/- MEFs have higher levels of the autophagy marker LC3b. Measurement of DNA content by flow cytometry revealed an increase in the percentage of Poldip2-/- cells in the G1 and G2/M phases of the cell cycle, accompanied by a decrease in the percentage of S-phase cells. Increases in p53 S20 and Sirt1 were observed in passage 2 Poldip2-/- MEFs. In passage 4/5 MEFs, Cdk1 and CyclinA2 are downregulated in Poldip2-/- cells, and these changes are reversed by transfection with SV40 large T-antigen, suggesting that Poldip2 may target the E2F pathway. In contrast, p21CIP1 is increased in passage 4/5 Poldip2-/- MEFs and its expression is unaffected by SV40 transfection. Overall, these results reveal that Poldip2 is an essential protein in development, and underline its importance in cell viability and proliferation. Because it affects the cell cycle, Poldip2 is a potential novel target for treating proliferative conditions such as cancer, atherosclerosis and restenosis.
- Published
- 2014
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39. Nox2-induced production of mitochondrial superoxide in angiotensin II-mediated endothelial oxidative stress and hypertension.
- Author
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Dikalov SI, Nazarewicz RR, Bikineyeva A, Hilenski L, Lassègue B, Griendling KK, Harrison DG, and Dikalova AE
- Subjects
- Angiotensin II pharmacology, Animals, CSK Tyrosine-Protein Kinase, Cyclic N-Oxides metabolism, Cyclic N-Oxides pharmacology, Cytoplasm metabolism, Disease Models, Animal, Electron Transport, Endothelial Cells metabolism, Gene Silencing, Humans, Hydrogen Peroxide metabolism, Hypertension physiopathology, Malates metabolism, Malates pharmacology, Membrane Glycoproteins genetics, Mice, Mice, Knockout, Mitochondria, Heart genetics, NADPH Oxidase 2, NADPH Oxidases genetics, Protein Isoforms, Protein Transport, RNA Interference, Reactive Oxygen Species metabolism, src-Family Kinases metabolism, Angiotensin II metabolism, Hypertension metabolism, Membrane Glycoproteins metabolism, Mitochondria, Heart metabolism, NADPH Oxidases metabolism, Oxidative Stress drug effects, Superoxides metabolism
- Abstract
Aims: Angiotensin II (AngII)-induced superoxide (O2(•-)) production by the NADPH oxidases and mitochondria has been implicated in the pathogenesis of endothelial dysfunction and hypertension. In this work, we investigated the specific molecular mechanisms responsible for the stimulation of mitochondrial O2(•-) and its downstream targets using cultured human aortic endothelial cells and a mouse model of AngII-induced hypertension., Results: Western blot analysis showed that Nox2 and Nox4 were present in the cytoplasm but not in the mitochondria. Depletion of Nox2, but not Nox1, Nox4, or Nox5, using siRNA inhibits AngII-induced O2(•-) production in both mitochondria and cytoplasm. Nox2 depletion in gp91phox knockout mice inhibited AngII-induced cellular and mitochondrial O2(•-) and attenuated hypertension. Inhibition of mitochondrial reverse electron transfer with malonate, malate, or rotenone attenuated AngII-induced cytoplasmic and mitochondrial O2(•-) production. Inhibition of the mitochondrial ATP-sensitive potassium channel (mitoK(+)ATP) with 5-hydroxydecanoic acid or specific PKCɛ peptide antagonist (EAVSLKPT) reduced AngII-induced H2O2 in isolated mitochondria and diminished cytoplasmic O2(•-). The mitoK(+)ATP agonist diazoxide increased mitochondrial O2(•-), cytoplasmic c-Src phosphorylation and cytoplasmic O2(•-) suggesting feed-forward regulation of cellular O2(•-) by mitochondrial reactive oxygen species (ROS). Treatment of AngII-infused mice with malate reduced blood pressure and enhanced the antihypertensive effect of mitoTEMPO. Mitochondria-targeted H2O2 scavenger mitoEbselen attenuated redox-dependent c-Src and inhibited AngII-induced cellular O2(•-), diminished aortic H2O2, and reduced blood pressure in hypertensive mice., Innovation and Conclusions: These studies show that Nox2 stimulates mitochondrial ROS by activating reverse electron transfer and both mitochondrial O2(•-) and reverse electron transfer may represent new pharmacological targets for the treatment of hypertension.
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- 2014
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40. Hydrogen peroxide regulates osteopontin expression through activation of transcriptional and translational pathways.
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Lyle AN, Remus EW, Fan AE, Lassègue B, Walter GA, Kiyosue A, Griendling KK, and Taylor WR
- Subjects
- Animals, Carrier Proteins genetics, Carrier Proteins metabolism, Cells, Cultured, Eukaryotic Initiation Factor-4E genetics, Eukaryotic Initiation Factor-4E metabolism, Gene Expression Regulation physiology, Intracellular Signaling Peptides and Proteins, Mutation, Myocytes, Smooth Muscle cytology, Osteopontin genetics, Phosphoproteins genetics, Phosphoproteins metabolism, Phosphorylation drug effects, Phosphorylation physiology, Polyribosomes genetics, Polyribosomes metabolism, Protein Biosynthesis physiology, Rats, Transcription, Genetic physiology, Gene Expression Regulation drug effects, Hydrogen Peroxide pharmacology, Myocytes, Smooth Muscle metabolism, Osteopontin biosynthesis, Oxidants pharmacology, Protein Biosynthesis drug effects, Transcription, Genetic drug effects
- Abstract
Recent in vivo studies establish that osteopontin (OPN) expression is hydrogen peroxide (H2O2)-dependent. However, the mechanisms by which H2O2 increases OPN expression remain poorly defined. OPN protein expression increased in an unusual biphasic pattern in response to H2O2. To investigate whether these increases were mediated through transcriptional and/or translational regulation of OPN, smooth muscle cells stimulated with 50 μM H2O2 were used as an in vitro cell system. Early protein increases at 6 h were not preceded by increased mRNA, whereas later increases (18 h) were, suggesting multiple mechanisms of regulation by H2O2. Polyribosomal fractionation assays established that early increases (6 h) in OPN expression were due to increased translation. This increase in translation occurred through phosphorylation of 4E-BP1 at the reactive oxygen species-sensitive Ser-65, which allowed for release and activation of eukaryotic initiation factor eIF4E and subsequent OPN translation. This early increase (6 h) in OPN was blunted in cells expressing a phospho-deficient 4E-BP1 mutant. H2O2 stimulation increased rat OPN promoter activity at 8 and 18 h, and promoter truncation studies established that promoter region -2284 to -795 is crucial for H2O2-dependent OPN transcription. ChIP studies determined that H2O2-dependent transcription is mediated by the reactive oxygen species-sensitive transcription factors NF-κB and AP-1. In conclusion, H2O2 stimulates OPN expression in a unique biphasic pattern, where early increases are translational and late increases are transcriptional.
- Published
- 2014
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41. Transforming growth factor β inhibits platelet derived growth factor-induced vascular smooth muscle cell proliferation via Akt-independent, Smad-mediated cyclin D1 downregulation.
- Author
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Martin-Garrido A, Williams HC, Lee M, Seidel-Rogol B, Ci X, Dong JT, Lassègue B, Martín AS, and Griendling KK
- Subjects
- Adolescent, Cell Proliferation drug effects, Cyclin D1 genetics, Gene Expression Regulation drug effects, Humans, Kruppel-Like Transcription Factors metabolism, Male, Proteolysis, Signal Transduction drug effects, Smad4 Protein metabolism, Transcription, Genetic, Cyclin D1 metabolism, Muscle, Smooth, Vascular cytology, Myocytes, Smooth Muscle drug effects, Myocytes, Smooth Muscle metabolism, Platelet-Derived Growth Factor pharmacology, Proto-Oncogene Proteins c-akt metabolism, Smad Proteins metabolism, Transforming Growth Factor beta pharmacology
- Abstract
In adult tissue, vascular smooth muscle cells (VSMCs) exist in a differentiated phenotype, which is defined by the expression of contractile proteins and lack of proliferation. After vascular injury, VSMC adopt a synthetic phenotype associated with proliferation, migration and matrix secretion. The transition between phenotypes is a consequence of the extracellular environment, and in particular, is regulated by agonists such as the pro-differentiating cytokine transforming growth factor β (TGFβ) and the pro-proliferative cytokine platelet derived growth factor (PDGF). In this study, we investigated the interplay between TGFβ and PDGF with respect to their ability to regulate VSMC proliferation. Stimulation of human aortic VSMC with TGFβ completely blocked proliferation induced by all isoforms of PDGF, as measured by DNA synthesis and total cell number. Mechanistically, PDGF-induced Cyclin D1 mRNA and protein expression was inhibited by TGFβ. TGFβ had no effect on PDGF activation of its receptor and ERK1/2, but inhibited Akt activation. However, constitutively active Akt did not reverse the inhibitory effect of TGFβ on Cyclin D1 expression even though inhibition of the proteasome blocked the effect of TGFβ. siRNA against Smad4 completely reversed the inhibitory effect of TGFβ on PDGF-induced Cyclin D1 expression and restored proliferation in response to PDGF. Moreover, siRNA against KLF5 prevented Cyclin D1 upregulation by PDGF and overexpression of KLF5 partially reversed TGFβ-induced inhibition of Cyclin D1 expression. Taken together, our results demonstrate that KLF5 is required for PDGF-induced Cyclin D1 expression, which is inhibited by TGFβ via a Smad dependent mechanism, resulting in arrest of VSMCs in the G1 phase of the cell cycle.
- Published
- 2013
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42. Polymerase delta interacting protein 2 sustains vascular structure and function.
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Sutliff RL, Hilenski LL, Amanso AM, Parastatidis I, Dikalova AE, Hansen L, Datla SR, Long JS, El-Ali AM, Joseph G, Gleason RL Jr, Taylor WR, Hart CM, Griendling KK, and Lassègue B
- Subjects
- Animals, Aorta drug effects, Aorta pathology, Aorta physiopathology, Aortic Aneurysm genetics, Aortic Aneurysm metabolism, Aortic Aneurysm pathology, Aortic Aneurysm physiopathology, Blood Pressure, Cells, Cultured, Collagen Type I metabolism, Cytochrome b Group metabolism, Dilatation, Pathologic, Disease Models, Animal, Dose-Response Relationship, Drug, Elastic Tissue metabolism, Extracellular Matrix metabolism, Gene Expression Regulation, Genotype, Male, Membrane Glycoproteins metabolism, Mice, Mice, Inbred C57BL, Mice, Knockout, Mitochondrial Proteins deficiency, Mitochondrial Proteins genetics, Myocytes, Smooth Muscle metabolism, NADH, NADPH Oxidoreductases metabolism, NADPH Oxidase 1, NADPH Oxidase 2, NADPH Oxidase 4, NADPH Oxidases metabolism, Nuclear Proteins deficiency, Nuclear Proteins genetics, Oxidants pharmacology, Phenotype, RNA, Messenger metabolism, Vascular Stiffness, Vasoconstrictor Agents pharmacology, Vasodilation, Aorta metabolism, Aortic Aneurysm prevention & control, Mitochondrial Proteins metabolism, Nuclear Proteins metabolism
- Abstract
Objective: On the basis of previous evidence that polymerase delta interacting protein 2 (Poldip2) increases reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (Nox4) activity in vascular smooth muscle cells, we hypothesized that in vivo knockdown of Poldip2 would inhibit reactive oxygen species production and alter vascular function., Approach and Results: Because homozygous Poldip2 deletion is lethal, Poldip2(+/-) mice were used. Poldip2 mRNA and protein levels were reduced by ≈50% in Poldip2(+/-) aorta, with no change in p22phox, Nox1, Nox2, and Nox4 mRNAs. NADPH oxidase activity was also inhibited in Poldip2(+/-) tissue. Isolated aortas from Poldip2(+/-) mice demonstrated impaired phenylephrine and potassium chloride-induced contractions, increased stiffness, and reduced compliance associated with disruption of elastic lamellae and excessive extracellular matrix deposition. Collagen I secretion was elevated in cultured vascular smooth muscle cells from Poldip2(+/-) mice and restored by H2O2 supplementation, suggesting that this novel function of Poldip2 is mediated by reactive oxygen species. Furthermore, Poldip2(+/-) mice were protected against aortic dilatation in a model of experimental aneurysm, an effect consistent with increased collagen secretion., Conclusions: Poldip2 knockdown reduces H2O2 production in vivo, leading to increases in extracellular matrix, greater vascular stiffness, and impaired agonist-mediated contraction. Thus, unaltered expression of Poldip2 is necessary for vascular integrity and function.
- Published
- 2013
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43. The bone morphogenic protein inhibitor, noggin, reduces glycemia and vascular inflammation in db/db mice.
- Author
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Koga M, Engberding N, Dikalova AE, Chang KH, Seidel-Rogol B, Long JS, Lassègue B, Jo H, and Griendling KK
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- Angiotensin II Type 1 Receptor Blockers pharmacology, Angiotensin II Type 1 Receptor Blockers therapeutic use, Animals, Blood Glucose drug effects, Blood Pressure drug effects, Blood Pressure physiology, Carrier Proteins pharmacology, Diabetes Mellitus, Type 2 physiopathology, Disease Models, Animal, Hyperglycemia physiopathology, Male, Mice, Mice, Mutant Strains, Reactive Oxygen Species metabolism, Tetrazoles pharmacology, Tetrazoles therapeutic use, Valine analogs & derivatives, Valine pharmacology, Valine therapeutic use, Valsartan, Vasculitis physiopathology, Bone Morphogenetic Proteins antagonists & inhibitors, Carrier Proteins therapeutic use, Diabetes Mellitus, Type 2 complications, Hyperglycemia prevention & control, Vasculitis prevention & control
- Abstract
Vascular diseases frequently accompany diabetes mellitus. Based on the current understanding of atherosclerosis as an inflammatory disorder of the vascular wall, it has been speculated that diabetes may accelerate atherosclerosis by inducing a proinflammatory milieu in the vasculature. ANG II and bone morphogenic proteins (BMPs) have been implicated in vascular inflammation. We evaluated the effect of angiotensin receptor blockade by valsartan and BMP inhibition by noggin on markers of vascular inflammation in a mouse model of diabetes. Noggin had no effect on blood pressure but decreased serum glucose levels, whereas valsartan significantly decreased blood pressure, but not serum glucose. Both inhibitors reduced reactive oxygen species production in the aorta. Additionally, noggin and valsartan diminish gene transcription and protein expression of various inflammatory molecules in the vascular wall. These observations indicate that although both inhibitors block superoxide production and have similar effects on inflammatory gene expression, glycemia and blood pressure may represent a secondary target differentially affected by noggin and valsartan. Our data clearly identify the BMP pathway as a potentially potent therapeutic target in diabetic inflammatory vascular disease.
- Published
- 2013
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44. Role of coronin 1B in PDGF-induced migration of vascular smooth muscle cells.
- Author
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Williams HC, San Martín A, Adamo CM, Seidel-Rogol B, Pounkova L, Datla SR, Lassègue B, Bear JE, and Griendling K
- Subjects
- Actin-Related Protein 2-3 Complex metabolism, Animals, Becaplermin, Carotid Artery Injuries metabolism, Carotid Artery Injuries pathology, Cells, Cultured, Disease Models, Animal, Humans, Kymography, Mice, Mice, Inbred C57BL, Microfilament Proteins genetics, Muscle, Smooth, Vascular pathology, Mutation, Myocytes, Smooth Muscle pathology, Neointima, Phosphorylation, Protein Kinase C-epsilon metabolism, Pseudopodia metabolism, RNA Interference, RNA, Messenger metabolism, Rats, Rats, Sprague-Dawley, Recombinant Proteins metabolism, Serine, Signal Transduction, Time Factors, Transfection, Cell Movement, Microfilament Proteins metabolism, Muscle, Smooth, Vascular metabolism, Myocytes, Smooth Muscle metabolism, Proto-Oncogene Proteins c-sis metabolism
- Abstract
Rationale: The type I subclass of coronins, a family of actin-binding proteins, regulates various actin-dependent cellular processes, including migration. However, the existence and role of coronins in vascular smooth muscle cell (VSMC) migration has yet to be determined., Objective: The goal of the present study was to define the mechanism by which coronins regulate platelet-derived growth factor (PDGF)-induced VSMC migration., Methods and Results: Coronin 1B (Coro1B) and 1C (Coro1C) were both found to be expressed in VSMCs at the mRNA and protein levels. Downregulation of Coro1B by siRNA increases PDGF-induced migration, while downregulation of Coro1C has no effect. We confirmed through kymograph analysis that the Coro1B-downregulation-mediated increase in migration is directly linked to increased lamellipodial protraction rate and protrusion distance in VSMC. In other cell types, coronins exert their effects on lamellipodia dynamics by an inhibitory interaction with the ARP2/3 complex, which is disrupted by the phosphorylation of Coro1B. We found that PDGF induces phosphorylation of Coro1B on serine-2 via PKCε, leading to a decrease in the interaction of Coro1B with the ARP2/3 complex. VSMCs transfected with a phosphodeficient S2A Coro1B mutant showed decreased migration in response to PDGF, suggesting that the phosphorylation of Coro1B is required for the promotion of migration by PDGF. In both the rat and mouse, Coro1B phosphorylation was increased in response to vessel injury in vivo., Conclusions: Our data suggest that phosphorylation of Coro1B and the subsequent reduced interaction with ARP2/3 complex participate in PDGF-induced VSMC migration, an important step in vascular lesion formation.
- Published
- 2012
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45. Biochemistry, physiology, and pathophysiology of NADPH oxidases in the cardiovascular system.
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Lassègue B, San Martín A, and Griendling KK
- Subjects
- Animals, Humans, Reactive Oxygen Species metabolism, Cardiovascular Diseases metabolism, Cardiovascular Diseases physiopathology, Cardiovascular Physiological Phenomena, Endothelium, Vascular enzymology, NADPH Oxidases physiology
- Abstract
The NADPH oxidase (Nox) enzymes are critical mediators of cardiovascular physiology and pathophysiology. These proteins are expressed in virtually all cardiovascular cells, and regulate such diverse functions as differentiation, proliferation, apoptosis, senescence, inflammatory responses and oxygen sensing. They target a number of important signaling molecules, including kinases, phosphatases, transcription factors, ion channels, and proteins that regulate the cytoskeleton. Nox enzymes have been implicated in many different cardiovascular pathologies: atherosclerosis, hypertension, cardiac hypertrophy and remodeling, angiogenesis and collateral formation, stroke, and heart failure. In this review, we discuss in detail the biochemistry of Nox enzymes expressed in the cardiovascular system (Nox1, 2, 4, and 5), their roles in cardiovascular cell biology, and their contributions to disease development.
- Published
- 2012
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46. Vascular smooth muscle insulin resistance, but not hypertrophic signaling, is independent of angiotensin II-induced IRS-1 phosphorylation by JNK.
- Author
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Hitomi H, Mehta PK, Taniyama Y, Lassègue B, Seidel-Rogol B, San Martin A, and Griendling KK
- Subjects
- Actins, Animals, Blotting, Western, Enzyme Inhibitors pharmacology, Hypertrophy metabolism, Immunoprecipitation, Muscle, Smooth, Vascular drug effects, Muscle, Smooth, Vascular pathology, Phosphorylation, Rats, Real-Time Polymerase Chain Reaction, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction drug effects, Transfection, Angiotensin II metabolism, Insulin Receptor Substrate Proteins metabolism, Insulin Resistance physiology, JNK Mitogen-Activated Protein Kinases metabolism, Muscle, Smooth, Vascular metabolism, Signal Transduction physiology
- Abstract
Angiotensin II (ANG II) has been implicated in the pathogenesis of diabetic micro- and macrovascular disease. In vascular smooth muscle cells (VSMCs), ANG II phosphorylates and degrades insulin receptor substrate-1 (IRS-1). While the pathway responsible for IRS-1 degradation in this system is unknown, c-Jun NH(2)-terminal kinase (JNK) has been linked with serine phosphorylation of IRS-1 and insulin resistance. We investigated the role of JNK in ANG II-induced IRS-1 phosphorylation, degradation, Akt activation, glucose uptake, and hypertrophic signaling, focusing on three IRS-1 phosphorylation sites: Ser302, Ser307, and Ser632. Maximal IRS-1 phosphorylation on Ser632 occurred at 5 min, on Ser307 at 30 min, and on Ser302 at 60 min. The JNK inhibitor SP600125 reduced ANG II-induced IRS-1 Ser307 phosphorylation (by 80%), IRS-1 Ser302 phosphorylation (by 70%), and IRS-1 Ser632 phosphorylation (by 50%). However, JNK inhibition had no effect on ANG II-mediated IRS-1 degradation, nor did it reverse the ANG II-induced decrease in Akt phosphorylation or glucose uptake. Transfection of VSMCs with mutants S307A, S302A, or S632A of IRS-1 did not block ANG II-mediated IRS-1 degradation. In contrast, JNK inhibition attenuated insulin-induced upregulation of collagen and smooth muscle α-actin in ANG II-pretreated cells. We conclude that phosphorylation of Ser307, Ser302, and Ser632 of IRS-1 is not involved in ANG II-mediated IRS-1 degradation, and that JNK alone does not mediate ANG II-stimulated IRS-1 degradation, but rather is responsible for the hypertrophic effects of insulin on smooth muscle.
- Published
- 2011
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47. Platelet-derived growth factor (PDGF) regulates Slingshot phosphatase activity via Nox1-dependent auto-dephosphorylation of serine 834 in vascular smooth muscle cells.
- Author
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Maheswaranathan M, Gole HKA, Fernandez I, Lassègue B, Griendling KK, and San Martín A
- Subjects
- 14-3-3 Proteins genetics, 14-3-3 Proteins metabolism, Amino Acid Motifs, Amino Acid Substitution, Animals, Enzyme Activation physiology, HEK293 Cells, Humans, Mice, Mice, Knockout, Mutation, Missense, NADH, NADPH Oxidoreductases genetics, NADPH Oxidase 1, Oxidation-Reduction, Phosphoprotein Phosphatases genetics, Phosphorylation physiology, Platelet-Derived Growth Factor genetics, Serine genetics, Serine metabolism, Muscle, Smooth, Vascular enzymology, Myocytes, Smooth Muscle enzymology, NADH, NADPH Oxidoreductases metabolism, Phosphoprotein Phosphatases metabolism, Platelet-Derived Growth Factor metabolism
- Abstract
Migration of vascular smooth muscle cells (VSMCs) contributes to vascular pathology. PDGF induces VSMC migration by a Nox1-based NADPH oxidase mediated mechanism. We have previously shown that PDGF-induced migration in VSMCs requires Slingshot-1L (SSH1L) phosphatase activity. In the present work, the mechanism of SSH1L activation by PDGF is further investigated. We identified a 14-3-3 consensus binding motif encompassing Ser-834 in SSH1L that is constitutively phosphorylated. PDGF induces SSH1L auto-dephosphorylation at Ser-834 in wild type (wt), but not in Nox1(-/y) cells. A SSH1L-S834A phospho-deficient mutant has significantly lower binding capacity for 14-3-3 when compared with the phospho-mimetic SSH1L-S834D mutant, and acts as a constitutively active phosphatase, lacking of PDGF-mediated regulation. Given that Nox1 produces reactive oxygen species, we evaluated their participation in this SSH1L activation mechanism. We found that H(2)O(2) activates SSH1L and this is accompanied by SSH1L/14-3-3 complex disruption and 14-3-3 oxidation in wt, but not in Nox1(-/y) cells. Together, these data demonstrate that PDGF activates SSH1L in VSMC by a mechanism that involves Nox1-mediated oxidation of 14-3-3 and Ser-834 SSH1L auto-dephosphorylation.
- Published
- 2011
- Full Text
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48. NADPH oxidase 4 mediates TGF-β-induced smooth muscle α-actin via p38MAPK and serum response factor.
- Author
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Martin-Garrido A, Brown DI, Lyle AN, Dikalova A, Seidel-Rogol B, Lassègue B, San Martín A, and Griendling KK
- Subjects
- Actins genetics, Aorta cytology, Aorta metabolism, Blotting, Western, Cells, Cultured, Fluorescent Antibody Technique, Humans, Imidazoles pharmacology, Luciferases metabolism, Myocytes, Smooth Muscle drug effects, Myocytes, Smooth Muscle metabolism, NADPH Oxidase 4, NADPH Oxidases antagonists & inhibitors, NADPH Oxidases genetics, Phosphorylation drug effects, Pyridines pharmacology, RNA, Messenger genetics, RNA, Small Interfering genetics, Reactive Oxygen Species metabolism, Reverse Transcriptase Polymerase Chain Reaction, Serum Response Factor genetics, Signal Transduction drug effects, Stress Fibers pathology, Transforming Growth Factor beta genetics, p38 Mitogen-Activated Protein Kinases antagonists & inhibitors, p38 Mitogen-Activated Protein Kinases genetics, Actins metabolism, NADPH Oxidases metabolism, Serum Response Factor metabolism, Stress Fibers metabolism, Transforming Growth Factor beta metabolism, p38 Mitogen-Activated Protein Kinases metabolism
- Abstract
In contrast to other cell types, vascular smooth muscle cells modify their phenotype in response to external signals. NADPH oxidase 4 (Nox4) is critical for maintenance of smooth muscle gene expression; however, the underlying mechanisms are incompletely characterized. Using smooth muscle α-actin (SMA) as a prototypical smooth muscle gene and transforming growth factor-β (TGF-β) as a differentiating agent, we examined Nox4-dependent signaling. TGF-β increases Nox4 expression and activity in human aortic smooth muscle cells (HASMC). Transfection of HASMC with siRNA against Nox4 (siNox4) abolishes TGF-β-induced SMA expression and stress fiber formation. siNox4 also significantly inhibits TGF-β-stimulated p38MAPK phosphorylation, as well as that of its substrate, mitogen-activated protein kinase-activated protein kinase-2. Moreover, the p38MAPK inhibitor SB-203580 nearly completely blocks the SMA increase induced by TGF-β. Inhibition of either p38MAPK or NADPH oxidase-derived reactive oxygen species impairs the TGF-β-induced phosphorylation of Ser103 on serum response factor (SRF) and reduces its transcriptional activity. Binding of SRF to myocardin-related transcription factor (MRTF) is also necessary, because downregulation of MRTF by siRNA abolishes TGF-β-induced SMA expression. Taken together, these data suggest that Nox4 regulates SMA expression via activation of a p38MAPK/SRF/MRTF pathway in response to TGF-β., (Copyright © 2010 Elsevier Inc. All rights reserved.)
- Published
- 2011
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49. NADPH oxidases: functions and pathologies in the vasculature.
- Author
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Lassègue B and Griendling KK
- Subjects
- Animals, Blood Vessels pathology, Cardiovascular Diseases pathology, Humans, Isoenzymes, Blood Vessels enzymology, Cardiovascular Diseases enzymology, NADPH Oxidases metabolism, Reactive Oxygen Species metabolism, Signal Transduction
- Abstract
Reactive oxygen species are ubiquitous signaling molecules in biological systems. Four members of the NADPH oxidase (Nox) enzyme family are important sources of reactive oxygen species in the vasculature: Nox1, Nox2, Nox4, and Nox5. Signaling cascades triggered by stresses, hormones, vasoactive agents, and cytokines control the expression and activity of these enzymes and of their regulatory subunits, among which p22phox, p47phox, Noxa1, and p67phox are present in blood vessels. Vascular Nox enzymes are also regulated by Rac, ClC-3, Poldip2, and protein disulfide isomerase. Multiple Nox subtypes, simultaneously present in different subcellular compartments, produce specific amounts of superoxide, some of which is rapidly converted to hydrogen peroxide. The identity and location of these reactive oxygen species, and of the enzymes that degrade them, determine their downstream signaling pathways. Nox enzymes participate in a broad array of cellular functions, including differentiation, fibrosis, growth, proliferation, apoptosis, cytoskeletal regulation, migration, and contraction. They are involved in vascular pathologies such as hypertension, restenosis, inflammation, atherosclerosis, and diabetes. As our understanding of the regulation of these oxidases progresses, so will our ability to alter their functions and associated pathologies.
- Published
- 2010
- Full Text
- View/download PDF
50. Poldip2, a novel regulator of Nox4 and cytoskeletal integrity in vascular smooth muscle cells.
- Author
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Lyle AN, Deshpande NN, Taniyama Y, Seidel-Rogol B, Pounkova L, Du P, Papaharalambus C, Lassègue B, and Griendling KK
- Subjects
- Animals, Cell Movement physiology, Cells, Cultured, Humans, Muscle, Smooth, Vascular cytology, NADPH Oxidase 4, Oxidation-Reduction, Rats, Reactive Oxygen Species metabolism, Saccharomyces cerevisiae, rhoA GTP-Binding Protein metabolism, Carrier Proteins metabolism, Cell Cycle Proteins metabolism, Cytoskeleton metabolism, Muscle, Smooth, Vascular metabolism, NADPH Oxidases metabolism, Nuclear Proteins metabolism
- Abstract
Rationale: NADPH oxidases (Noxes) regulate vascular physiology and contribute to the pathogenesis of vascular disease. In vascular smooth muscle cells (VSMCs), the interactions of individual Nox homologs with regulatory proteins are poorly defined., Objective: The objective of this study was to identify novel NADPH oxidase regulatory proteins., Methods and Results: Using a yeast 2-hybrid screen, we identified a novel p22phox binding partner, Poldip2, and demonstrated that it associates with p22phox, NADPH oxidase (Nox)1, and Nox4 and colocalizes with p22phox at sites of Nox4 localization. Poldip2 increases Nox4 enzymatic activity by 3-fold and positively regulates basal reactive oxygen species production in VSMCs (O2(.-): 86.3+/-15.6% increase; H2O2: 40.7+/-4.5% increase). Overexpression of Poldip2 activates Rho (180.2+/-24.8% increase), strengthens focal adhesions, and increases stress fiber formation. These phenotypic changes are blocked by dominant negative Rho. In contrast, depletion of either Poldip2 or Nox4 results in a loss of these structures, which is rescued by adding back active Rho. Cell migration, which requires dynamic cytoskeletal remodeling, is impaired by either excess (70.1+/-14.7% decrease) or insufficient Poldip2 (63.5+/-5.9% decrease)., Conclusions: These results suggest that Poldip2 associates with p22phox to activate Nox4, leading to regulation of focal adhesion turnover and VSMC migration, thus linking reactive oxygen species production and cytoskeletal remodeling. Poldip2 may be a novel therapeutic target for vascular pathologies with a significant VSMC migratory component, such as restenosis and atherosclerosis.
- Published
- 2009
- Full Text
- View/download PDF
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