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1.
Arterioscler Thromb Vasc Biol ; 38(10): 2423-2434, 2018 10.
Article in English | MEDLINE | ID: mdl-30354218

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 (H2O2) 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 H2O2 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.


Subject(s)
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 , 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
2.
J Neuroinflammation ; 15(1): 45, 2018 Feb 17.
Article in English | MEDLINE | ID: mdl-29452577

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.


Subject(s)
Blood-Brain Barrier/metabolism , Brain Ischemia/metabolism , Brain Ischemia/prevention & control , Capillary Permeability/physiology , Mitochondrial Proteins/deficiency , Neuroprotection/physiology , Nuclear Proteins/deficiency , Animals , Brain/diagnostic imaging , Brain/metabolism , Brain Ischemia/diagnostic imaging , Cells, Cultured , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic
3.
Am J Physiol Heart Circ Physiol ; 307(7): H945-57, 2014 Oct 01.
Article in English | MEDLINE | ID: mdl-25063792

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.


Subject(s)
Carrier Proteins/metabolism , Cell Movement , Focal Adhesions/metabolism , Myocytes, Smooth Muscle/metabolism , 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
4.
Arterioscler Thromb Vasc Biol ; 33(9): 2154-61, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23825363

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.


Subject(s)
Aorta/metabolism , Aortic Aneurysm/prevention & control , Mitochondrial Proteins/metabolism , Nuclear Proteins/metabolism , 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
5.
Free Radic Biol Med ; 37(10): 1542-9, 2004 Nov 15.
Article in English | MEDLINE | ID: mdl-15477006

ABSTRACT

The vascular NAD(P)H oxidases constitute important sources of ROS in the vessel wall and have been implicated in vascular disease. Vascular smooth muscle cells (VSMCs) from conduit arteries express two gp91phox homologs, Nox1 and Nox4, of which Nox1 is agonist-sensitive. Because p22phox has been shown to be functionally important in vascular cells stimulated with vasoactive hormones, the relationship of Nox1 and p22phox was investigated in VSMCs from rat and human aortas. Coimmunoprecipitation studies demonstrated that p22phox and hemagglutinin-tagged Nox1 associate in unstimulated VSMCs. These findings were confirmed by confocal microscopy, showing colocalization of the two proteins in their native states in the plasma membrane and submembrane areas of the cell. NADPH-driven superoxide production, as measured by electron spin resonance using 1-hydroxy-3-carboxypyrrolidine as a spin probe, is dependent on the coexpression of both subunits, suggesting the importance of the association for the functional integrity of the enzyme. These results indicate that in contrast to the neutrophil enzyme, VSMCs can use Nox1 rather than gp91phox as a catalytic center in the p22phox-based oxidase and that these two proteins are preassembled at or near the plasma membrane and submembrane vesicular structures in unstimulated cells.


Subject(s)
Aorta/metabolism , Membrane Transport Proteins/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , NADH, NADPH Oxidoreductases/metabolism , NADPH Dehydrogenase/metabolism , Phosphoproteins/metabolism , Animals , Cell Membrane/metabolism , Cells, Cultured , Electron Spin Resonance Spectroscopy , NADPH Oxidase 1 , NADPH Oxidases , Protein Binding , Rats
6.
Arterioscler Thromb Vasc Biol ; 24(7): 1223-8, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15142861

ABSTRACT

OBJECTIVE: Microtubules are important in signal transduction temporal-spatial organization. Full expression of angiotensin II (Ang II) signaling in vascular smooth muscle cells (VSMCs) is dependent on the reactive oxygen species (ROS) derived from nicotinamide-adenine dinucleotide phosphate (NAD(P)H) oxidase and the dynamic association of the Ang II type 1 receptor (AT1R) with caveolae/lipid rafts. Translocation of the small GTPase Rac1 to the plasma membrane is an essential step for activation of NAD(P)H oxidase; however, its precise localization in the plasma membrane after agonist stimulation and how it is targeted are unknown. We hypothesized that microtubules are involved in regulating multiphasic Ang II signaling events in VSMC. METHODS AND RESULTS: We show that Ang II promotes Rac1 and AT1R trafficking into caveolae/lipid rafts, which is blocked by disruption of microtubules with nocodazole. As a consequence, nocodazole significantly inhibits Ang II-stimulated H2O2 production, its downstream ROS-dependent epidermal growth factor receptor transactivation, Akt phosphorylation, and vascular hypertrophy without affecting Rac1 activation or ROS-independent extracellular signal-regulated kinase 1/2 phosphorylation. CONCLUSIONS: These results suggest that proper Rac1 and AT1R trafficking into caveolae/lipid rafts requires the integrity of microtubules and provide insight into the essential role of microtubules for the spatial-temporal organization of ROS-dependent and caveolae/lipid rafts-dependent AT(1)R signaling linked to vascular hypertrophy.


Subject(s)
Angiotensin II/pharmacology , Caveolae/metabolism , Membrane Microdomains/metabolism , Microtubules/physiology , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/drug effects , Receptor, Angiotensin, Type 1/metabolism , rac1 GTP-Binding Protein/metabolism , Animals , Aorta/cytology , Epidermal Growth Factor/pharmacology , ErbB Receptors/metabolism , Hydrogen Peroxide/pharmacology , Hypertrophy , Microtubules/drug effects , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/ultrastructure , Nocodazole/pharmacology , Oxidation-Reduction , Oxidative Stress , Phosphorylation/drug effects , Protein Processing, Post-Translational/drug effects , Protein Serine-Threonine Kinases/metabolism , Protein Transport/drug effects , Protein Transport/physiology , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Rats , Reactive Oxygen Species
7.
Arterioscler Thromb Vasc Biol ; 24(4): 677-83, 2004 Apr.
Article in English | MEDLINE | ID: mdl-14670934

ABSTRACT

OBJECTIVE: Reactive oxygen species (ROS) that act as signaling molecules in vascular smooth muscle cells (VSMC) and contribute to growth, hypertrophy, and migration in atherogenesis are produced by multi-subunit NAD(P)H oxidases. Nox1 and Nox4, two homologues to the phagocytic NAD(P)H subunit gp91phox, both generate ROS in VSMC but differ in their response to growth factors. We hypothesize that the opposing functions of Nox1 and Nox4 are reflected in their differential subcellular locations. METHODS AND RESULTS: We used immunofluorescence to visualize the NAD(P)H subunits Nox1, Nox4, and p22phox in cultured rat and human VSMC. Optical sectioning using confocal microscopy showed that Nox1 is co-localized with caveolin in punctate patches on the surface and along the cellular margins, whereas Nox4 is co-localized with vinculin in focal adhesions. These immunocytochemical distributions are supported by membrane fractionation experiments. Interestingly, p22phox, a membrane subunit that interacts with the Nox proteins, is found in surface labeling and in focal adhesions in patterns similar to Nox1 and Nox4, respectively. CONCLUSIONS: The differential roles of Nox1 and Nox4 in VSMC may be correlated with their differential compartmentalization in specific signaling domains in the membrane and focal adhesions.


Subject(s)
Caveolae/enzymology , Focal Adhesions/enzymology , Muscle, Smooth, Vascular/enzymology , Myocytes, Smooth Muscle/enzymology , NADH, NADPH Oxidoreductases/analysis , NADPH Oxidases/analysis , Animals , Caveolin 1 , Caveolins/analysis , Cell Division , Cell Fractionation , Cells, Cultured/enzymology , Cells, Cultured/ultrastructure , Cellular Senescence , Cytoskeleton/metabolism , Humans , Macromolecular Substances , Male , Membrane Transport Proteins/analysis , Microscopy, Confocal , Microscopy, Fluorescence , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/ultrastructure , NADH, NADPH Oxidoreductases/physiology , NADPH Dehydrogenase/analysis , NADPH Oxidase 1 , NADPH Oxidase 4 , NADPH Oxidases/physiology , Oxidation-Reduction , Phosphoproteins/analysis , Protein Subunits , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Vinculin/analysis
8.
Arterioscler Thromb Vasc Biol ; 22(1): 21-7, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11788456

ABSTRACT

Restenosis, a frequent complication of coronary angioplasty, is associated with increased superoxide (O2*(-)) production. Although the molecular identity of the responsible oxidase is unclear, an NAD(P)H oxidase appears to be involved. In smooth muscle, p22phox and 2 homologues of gp91phox, nox1 and nox4, are expressed, whereas fibroblasts contain gp91phox. To begin investigating the possibility that these oxidase components might contribute to the increased O2*(-) that accompanies neointimal formation, we measured their expression after balloon injury of the rat carotid artery. The increase in O2*(-) production 3 to 15 days after surgery was not due to inflammatory cell infiltration but appeared to be derived from medial and neointimal smooth muscle cells and adventitial fibroblasts. Nox1 and p22phox mRNAs were increased 2.7- and 3.6-fold, respectively, at day 3 after injury and remained elevated for 15 days. gp91Phox was increased 7 to 15 days after injury, and nox4 expression was increased 2-fold, but only at day 15 after surgery. These results confirm and extend our previous in vitro data and suggest that in the vasculature, the nox-based NAD(P)H oxidases serve different functions. This dynamic regulation of oxidase components may be critical to smooth muscle phenotypic modulation in restenosis and atherosclerosis.


Subject(s)
Carotid Artery Injuries/metabolism , Membrane Transport Proteins , Muscle, Smooth, Vascular/metabolism , NADH, NADPH Oxidoreductases/metabolism , NADPH Dehydrogenase/metabolism , NADPH Oxidases/metabolism , Phosphoproteins/metabolism , Superoxides/metabolism , Animals , Catheterization/adverse effects , Cell Division , Constriction, Pathologic/metabolism , Fibroblasts/metabolism , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/injuries , NADPH Oxidase 1 , NADPH Oxidase 4 , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Time Factors , Tunica Intima/cytology , Tunica Intima/metabolism
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