Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0022116 (ischemia)
 91,303 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Vascular endothelial growth factor (VEGF) has been proposed to be among the candidate factors with the most potential to play a role in ischemia-induced collateral vessel formation. Recently, we found that VEGF activated the mitogen-activated protein kinase cascade in cultured rat cardiac myocytes. To elucidate how VEGF affects adhesive interaction of cardiac myocytes with the extracellular matrix (ECM), one of the important cell functions, we investigated the molecular mechanism of activation of focal adhesion-related proteins, especially focal adhesion kinase (p125(FAK)), in cultured rat cardiac myocytes. We found that the 2 VEGF receptors, KDR/Flk-1 and Flt-1, were expressed in cardiac myocytes and that KDR/Flk-1 was significantly tyrosine phosphorylated on VEGF stimulation. VEGF induced tyrosine phosphorylation and activation of p125(FAK) as well as tyrosine phosphorylation of paxillin; this was accompanied by subcellular translocation of p125(FAK) from perinuclear sites to the focal adhesions. This VEGF-induced activation of p125(FAK) was inhibited partially by the tyrosine kinase inhibitors genistein and tyrphostin. Activation of p125(FAK) was accompanied by its increased association with adapter proteins GRB2, Shc, and nonreceptor type tyrosine kinase p60(c-src). Furthermore, we confirmed that VEGF induced a significant increase in adhesive interaction between cardiac myocytes and ECM using an electric cell-substrate impedance sensor. These results strongly suggest that p125(FAK) is one of the most important components in VEGF-induced signaling in cardiac myocytes, playing a critical role in adhesive interaction between cardiac myocytes and ECM.
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PMID:Vascular endothelial growth factor induces activation and subcellular translocation of focal adhesion kinase (p125FAK) in cultured rat cardiac myocytes. 1034 94

Actin cytoskeletal disruption is a hallmark of ischemic injury and ATP depletion in a number of cell types, including renal epithelial cells. We manipulated Rho GTPase signaling by transfection and microinjection in LLC-PK proximal tubule epithelial cells and observed actin cytoskeletal organization following ATP depletion or recovery by confocal microscopy and quantitative image analysis. ATP depletion resulted in disruption of stress fibers, cortical F-actin, and apical actin bundles. Constitutively active RhoV14 prevented disruption of stress fibers and cortical F-actin during ATP depletion and enhanced the rate of stress fiber reassembly during recovery. Conversely, the Rho inhibitor C3 or dominant negative RhoN19 prevented recovery of F-actin assemblies upon repletion. Actin bundles in the apical microvilli and cytosolic F-actin were not affected by Rho signaling. Assembly of vinculin and paxillin into focal adhesions was disrupted by ATP depletion, and constitutively active RhoV14, although protecting stress fibers from disassembly, did not prevent dispersion of vinculin and paxillin, resulting in uncoupling of stress fiber and focal adhesion assembly. We propose that ATP depletion causes Rho inactivation during ischemia and that recovery of normal cellular architecture and function requires Rho.
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PMID:Rho controls actin cytoskeletal assembly in renal epithelial cells during ATP depletion and recovery. 1036 94

Interaction between neutrophils and endothelial cells is one of the first steps in the functional response of polymorphonuclear neutrophils (PMN), and is necessary for their migration toward damaged tissues. PMN activation, leading to their adhesion to and migration between endothelial cells, is part of a complex phenomenon that can be altered in pathological situations such as the ischemia-reperfusion syndrome, in which large numbers of PMN are recruited to the tissue and release reactive oxygen species (ROS) near the vessel wall. ROS have been implicated in the pathogenesis of various inflammatory diseases. The increased adhesion of PMN to ROS-stimulated endothelial cells involves an increase in tyrosine phosphorylation of a tyrosine kinase focal adhesion kinase (p125FAK) and several cytoskeleton proteins, including paxillin and p130 cas. We examined the role of glutathione (GSH) in the regulation of this adhesion phenomenon and in the increased tyrosine phosphorylation induced by ROS. For this purpose we used anethole dithiolthione (ADT), which increases the glutathione synthesis by activating gamma-glutamyl-cysteine synthetase. We found that ADT reduced both PMN adhesion to ROS-stimulated human umbilical vein endothelial cells (HUVEC) and tyrosine phosphorylation of p125FAK and paxillin. ADT increased redox status by increasing intracellular GSH content in oxidized cells. These results show that GSH can reverse the effect of oxidation on tyrosine kinase activation and phosphorylation, and thus plays an important role in cell signaling. They also confirm the antioxidant activity of ADT.
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PMID:Anethole dithiolethione regulates oxidant-induced tyrosine kinase activation in endothelial cells. 1121 83

Anaerobic mitochondrial metabolism of alpha-ketoglutarate and aspartate or alpha-ketoglutarate and malate can prevent and reverse severe mitochondrial dysfunction during reoxygenation after 60 minutes of hypoxia in kidney proximal tubules.(34) The present studies demonstrate that, during hypoxia, paxillin, focal adhesion kinase, and p130(cas) migrated faster by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their phosphotyrosine (pY) content decreased to approximately 5% of that in oxygenated tubules without changes in total protein, and the normally basal immunostaining of beta1 and alpha6 integrin subunits, pY, and paxillin was lost or markedly decreased. During reoxygenation without supplemental substrates, recovery of pY and basal localization of the focal adhesion proteins was poor. alpha-Ketoglutarate and aspartate, which maintained slightly higher levels of ATP during hypoxia, also maintained 2.5-fold higher levels of pY during this period, and promoted full recovery of pY content and basal localization of focal adhesion proteins during subsequent reoxygenation. Similarly complete recovery was made possible by provision of alpha-ketoglutarate and aspartate or alpha-ketoglutarate and malate only during reoxygenation. These data emphasize the importance of very low energy thresholds for maintaining the integrity of key structural and biochemical components required for cellular survival and reaffirm the value of approaches aimed at conserving or generating energy in cells injured by hypoxia or ischemia.
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PMID:Energetic determinants of tyrosine phosphorylation of focal adhesion proteins during hypoxia/reoxygenation of kidney proximal tubules. 1139 93

Disruption of cell contact sites during ischemia contributes to the loss of organ function in acute renal failure. Because prior heat stress protects cell contact sites in ATP-depleted renal epithelial cells in vitro, we hypothesized that heat shock protein 72 (HSP72), the major inducible cytoprotectant in mammalian cells, interacts with protein kinases that regulate cell-cell and cell-matrix interactions. ATP depletion increased the content of Tyr(416) Src, the activated form of this kinase. c-Src activation was associated with an increase in the tyrosine phosphorylation state of beta-catenin, paxillin, and vinculin, three c-Src substrate proteins that localize to and regulate cell contact sites. Prior heat stress inhibited c-Src activation and decreased the degree of tyrosine phosphorylation of all three Src substrates during ATP depletion and/or early recovery. HSP72 coimmunoprecipitated with c-Src only in cells subjected to heat stress. ATP depletion markedly increased the interaction between HSP72 and c-Src, supporting the hypothesis that HSP72 regulates Src kinase activity. These results suggest that alterations in the tyrosine phosphorylation state of proteins located at the cell-cell and cell-matrix interface mediate, at least in part, the functional state of these structures during ATP depletion and may be modulated by interactions between HSP72 and c-Src.
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PMID:c-Src and HSP72 interact in ATP-depleted renal epithelial cells. 1160 Apr 31

The transition from reversible to irreversible ischemic injury (ischemia-reperfusion, I/R) occurs coincident with the loss of vinculin, a cytoskeletal protein involved in the attachment of the myofibrils to the sarcolemmal membrane. If the loss of vinculin were critical to the development of I/R, then increased levels of vinculin would be predicted to delay the onset of irreversible injury assuming that the protein is functional and localized to the proper subcellular site. The present study determined whether increased expression of vinculin, specifically in the cytoskeletal compartment, would provide protection from I/R injury. Neonatal rat myocytes were cultured and infected with a newly created replication-deficient adenovirus driving the expression of vinculin. I/R was induced with chemical inhibitors of glycolysis and mitochondrial respiration. Irreversible cell injury was assessed with lactate dehydrogenase (LDH) release. Virus-infected myocytes expressed significantly more vinculin in the cytoskeletal fraction and increased the expression of paxillin but sustained the same amount of injury in response to simulated I/R as control cells (n = 4; P = not significant, paired t-test). Hypothermic I/R (ischemia at 25 degrees C) resulted in a significant reduction in LDH release (P </= 0.02; n = 4). Virus-mediated overexpression of vinculin does not appear to represent a rational approach to overcoming I/R injury.
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PMID:Effect of increased expression of cytoskeletal protein vinculin on ischemia-reperfusion injury in ventricular myocytes. 1257 17

Restoration of proximal tubular cell (PTC) integrity and function after ischemic injury involves cell proliferation and migration. Hypoxia is a known stimulus for PTC TGF-beta1 synthesis. This study examines the effect of TGF-beta1 on PTC migration. A model of PTC injury was used consisting of mechanically wounding a monolayer of HK2 cells followed by repopulation of the denuded area by time lapse photomicroscopy. Repopulation was the result of cell migration but not proliferation. Addition of TGF-beta1 led to a marked inhibition of cell migration increased expression of paxillin and vincullin and their incorporation into dense focal adhesion plaques. This was associated with increased association of focal adhesion components with the f-actin cytoskeleton. There was also increased beta3 integrin expression and increased synthesis of the matrix component fibronectin. The effect on migration and focal adhesion reorganisation was abrogated by inhibitors of the RhoA downstream target ROCK, suggesting that signaling events resulting from altered beta3 integrin expression initiate the TGF-beta1 response. These results suggest that, by inhibition of cell migration, increased expression of TGF-beta1 after ischemia delays recovery of proximal tubule structure and function. We speculate that this may contribute to permanent alteration in renal tubular function after severe ischemic injury.
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PMID:TGF-beta1-mediated inhibition of HK-2 cell migration. 1259 98

Focal adhesion kinase (FAK) is a 125 kDa protein tyrosine kinase (PTK) associated with focal adhesion in many cells, which plays a major role in the integrity of cytoskeletal structure. Reactive oxygen species produced during ischemia and reperfusion injury has been found to be an important mediator of signal transduction process. We found that low dose H2O2 induced increased FAK production in pulmonary microvascular endothelial cells, which could be blocked by cycloheximide (CHX), a protein synthesis inhibitor. Pulmonary endothelial cells were cultured on DMEM medium till 100% confluent. H2O2 was added at 100 uM for 30 min. The cells were collected and lysed, then immuno-blotted with anti-FAK antibody. After 30 min treatment, we found a 30%+/-6% (N=5) increase of FAK in H2O2 treated endothelial cells. This increase could be blocked by pretreatment of cells with CHX at 5 ug/ml for 60 min. In both groups, increased phosphorylation of ERK was observed. Immuno-fluorescence revealed increased staining of FAK in the peri-nuclear region of the H2O2 treated endothelial cells. These findings suggest that H2O2 activated MAP kinase pathway leading to increased FAK production at the protein level. FAK is a 125 kDa PTK associated with focal adhesion in many cells, and it plays a major role in the integrity of cytoskeletal structure. FAK is discretely localized to focal adhesions via its C-terminal focal adhesion-targeting (FAT) sequence. FAK is regulated by integrin-dependent cell adhesion and can control tyrosine phosphorylation of downstream substrates, like paxillin. The reactive oxygen species produced during ischemia and reperfusion injury has been found to be an important mediator of the signal transduction process. Although the signaling pathways leading to hydrogen peroxide induced endothelial monolayer permeability remain ambiguous, cytoskeletal proteins are known to be essential for maintaining endothelial integrity and regulating solute flux through the monolayer. Recent evidence has shown that H2O2 stimulates cytoskeleton reorganization, cell growth/proliferation, and DNA synthesis in various cells. In our previous study, we found a significantly increased amount of FAK in endothelial cells treated with low doses of H2O2. Mitogen-activated protein (MAP) kinases are a group of 30- to 110-kDa serine/threonine kinases. MAPKs belong to the group of kinases that are rapidly activated in response to growth factor stimulation. This family of MAPKs includes ERK, and ERK2. The activated MAPK can translocate to the nucleus where it can regulate transcription factors. Activation of p44 and p42 extracellular signal-regulated protein kinases (ERK1 and ERK2) is an important step in the cascade leading to cell growth and proliferation. In order to determine the mechanism of increased FAK production, we investigated the relationship of FAK production and ERK activation.
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PMID:Reactive oxygen species increased focal adhesion kinase production in pulmonary microvascular endothelial cells. 1563 12

Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury in both native kidneys and renal allografts. Disruption of the actin cytoskeleton is a key event with multiple repercussions on cell adhesion and function during IRI. However, receptors involved in regulating cytoskeletal repair following injury have not been identified. In an in vivo model of renal IRI, we used multiprobe RNase protection assay to examine the expression of Eph receptor tyrosine kinases, key regulators of actin dynamics in embryonic development. We found that one receptor in particular, EphA2, was strongly upregulated in the kidney following IRI. Ephrins, the cell-bound ligands of Eph receptors, were also strongly expressed. In cultured renal tubular cells, diverse injurious stimuli mimicking IRI also resulted in upregulation of EphA2 protein expression. Upregulation of EphA2 was inhibited by the Src kinase inhibitor PP2. Conversely, overexpression of Src kinases strongly enhanced the expression of endogenous EphA2 as well as the activity of a human EphA2 promoter construct. Activation of the Erk pathway was necessary, but not sufficient for full induction of EphA2 upreglation by Src kinases. Stimulation of renal tubular epithelial cells with the EphA2 ligand ephrin-A1 caused tyrosine phosphorylation of endogenous EphA2, paxillin, and an unidentified approximately 65-kDa protein and resulted in increased cortical F-actin staining. In summary, under in vitro conditions mimicking IRI, EphA2 expression is strongly upregulated through a Src kinase-dependent pathway. Interactions between upregulated EphA2 and its ephrin ligands may provide critical cell contact-dependent, bidirectional cues for cytoskeletal repair in renal IRI.
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PMID:Upregulation of EphA2 during in vivo and in vitro renal ischemia-reperfusion injury: role of Src kinases. 1673 61

Sublethal renal ischemia induces tubular epithelium damage and kidney dysfunction. Using NRK-52E rat proximal tubular epithelial cells, we have established an in vitro model, which includes oxygen and nutrients deprivation, to study the proximal epithelial cell response to ischemia. By means of this system, we demonstrate that confluent NRK-52E cells lose monolayer integrity and detach from collagen IV due to: (i) actin cytoskeleton reorganization; (ii) Rac1 and RhoA activity alterations; (iii) Adherens junctions (AJ) and Tight junctions (TJ) disruption, involving redistribution but not degradation of E-cadherin, beta-catenin and ZO-1; (iv) focal adhesion complexes (FAC) disassembly, entangled by mislocalization of paxillin and FAK dephosphorylation. Reactive oxygen species (ROS) are generated during the deprivation phase and rapidly balanced at recovery involving MnSOD induction, among others. The use of antioxidants (NAC) prevented FAC disassembly by blocking paxillin redistribution and FAK dephosphorylation, without abrogating AJ or TJ disruption. In spite of this, NAC did not show any protective effect on cell detachment. H(2)O(2), as a pro-oxidant treatment, supported the contribution of ROS in tubular epithelial cell-matrix but not cell-cell adhesion alterations. In conclusion, ROS-mediated FAC disassembly was not sufficient for the proximal epithelial cell shedding in response to sublethal ischemia, which also requires intercellular adhesion disruption.
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PMID:Requirements for proximal tubule epithelial cell detachment in response to ischemia: role of oxidative stress. 1702 98


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