Classification of injuries observed in functional classes of self-injurious behaviour.

BACKGROUND: Limited research has examined how the functions of self-injurious behaviour (SIB) relate to the production of injuries and the location, type or severity of those injuries. METHODS: Clinical and medical records were coded for 64 individuals hospitalised for SIB. When injuries were present, the physical properties of SIB and injuries were assessed across groups of individuals with automatically and socially maintained SIB. RESULTS: Injuries were observed for 35 of the individuals who engaged in SIB. Individuals who engaged in a single form of SIB were more likely to have injuries (P < .05). Individuals with SIB maintained by automatic reinforcement had significantly more severe injuries to the head than those in the social group (q < .05, P = .0132, H = 12.54). CONCLUSION: Although results are preliminary, the results provide evidence that the function of SIB may influence the severity and location of injuries produced.

KATP channels are an important component of the shear-sensing mechanism in the pulmonary microvasculature.

OBJECTIVE: To investigate the role of a KATP channel in sensing shear, specifically its cessation, in the endothelial cells of the pulmonary microvasculature. METHODS: Endothelial cells isolated from the pulmonary microvasculature of wild-type and KATP channel knockout (KIR6.2-/-) mice were either statically cultured (non-flow-adapted) or kept under flow (flow-adapted) and the KIR currents in these cells were monitored by whole-cell patch-clamp technique during flow and its cessation. Membrane potential changes, generation of reactive oxygen species (ROS), and Ca2+ influx with flow cessation were evaluated by the use of fluorescent dyes. Lungs isolated from wild-type mice were imaged to visualize ROS generation in the subpleural endothelium. RESULTS: By patch-clamp analysis, reduction in the KIR current with cessation of flow occurred only in wild-type cells that were flow-adapted and not in flow-adapted KIR6.2-/- cells. Similar observations were made using changes in bisoxonol fluorescence as an index of cell membrane potential. Generation of ROS and Ca2+ influx that follow membrane depolarization were significantly lower in statically cultured and in KIR6.2-/- cells as compared to flow-adapted wild-type cells. Imaging of subpleural endothelial cells of the whole lung showed that the KATP antagonist glyburide caused the production of ROS in the absence of flow cessation. CONCLUSIONS: The responses to stop of flow (viz. membrane depolarization, KIR currents, ROS, Ca2+) were significantly altered with knockout of KATP channels, which indicates that this channel is an important component of the pulmonary endothelial response to abrupt loss of shear stress.

Reactive oxygen species and cell signaling with lung ischemia.

In summary, our studies, utilizing the intact lung and several in vitro models, have shown a characteristic response of flow-adapted endothelial cells to ischemia. We believe that this effect represents a response to decreased shear stress since it is unrelated to cellular oxygenation. The response is characterized by endothelial cell depolarization, followed by activation of the membrane-bound NADPH oxidase with generation of ROS, cell signaling, activation of transcription factors, and increased cell division. We postulate that the physiologic role of this response is an attempt to restore blood flow through vasodilation and the repair or genesis of blood vessels.

Activation of the antioxidant enzyme 1-CYS peroxiredoxin requires glutathionylation mediated by heterodimerization with pi GST.

1-cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin superfamily, can protect cells against membrane oxidation through glutathione (GSH)-dependent reduction of phospholipid hydroperoxides to corresponding alcohols. However, purified native or recombinant enzyme in vitro generally lacks GSH peroxidase (GPx) activity because of oxidation of its single conserved cysteine. Reduction of the resultant oxidized cysteine is difficult because of its protected location within the homodimer formed by the oxidized protein monomers. Partial purification of 1-cysPrx from bovine lung revealed the presence of pi GST in an active preparation, while purification to homogeneity yielded enzyme that inactivated with time. We show that heterodimerization of 1-cysPrx with GSH-saturated pi GST results in glutathionylation of the oxidized cysteine in 1-cysPrx followed by subsequent spontaneous reduction of the mixed disulfide and restoration of enzymatic activity. Maximum activation of 1-cysPrx occurred with a 1:1 molar ratio of GSH-saturated pi GST and a 2:1 molar ratio of GSH to 1-cysPrx. Liposome-mediated delivery of oxidized recombinant enzyme into NCI-H441 cells that lack 1-cysPrx but express pi GST resulted in 1-cysPrx activation, whereas activation in MCF7 cells required co-delivery of pi GST. Our data indicate a physiological mechanism for glutathionylation of the oxidized catalytic cysteine of 1-cysPrx by its heterodimerization with pi GST followed by its GSH-mediated reduction and enzyme activation.

Differential effects of transforming growth factor on cell cycle regulatory molecules in human myeloid leukemia cells.

In this report we have studied the mechanism by which Transforming Growth Factor beta (TGF beta) inhibits growth of human myeloid leukemia cell lines. TGF beta 1 arrested cells in G1 phase and significantly downregulated the expression of cyclin D2, cyclin D3, cdk4, cyclin A, and cdk2. The downregulation of the molecules resulted in approximately 50-90% decrease of the molecule-dependent kinase activity, varying with each molecule. Although treatment of cells with TGF beta 1 up-regulated accumulation of p27(kip1) in both nucleus and cytoplasm, the association of the p27(kip1) with cdk2, cyclin A, cyclin D2, cyclin D3, and cdk4 was markedly down-regulated, suggesting that p27(kip1) is not responsible for the downregulation of the kinase activity. In contrast, TGF beta 1 upregulated cyclin E-associated p27(kip1) with no effect on the expression of cyclin E. p27(kip1)-immunodepletion upregulated cyclin E-dependent kinase activity by more than 10-fold in TGF beta 1-treated cells but not in proliferating cells; whereas immunodepletion of p27(kip1) from cdk2-immunoprecipitates markedly downregulated cdk2 kinase activity in the lysates extracted from both proliferating and TGF beta-treated cells. Consistent with this observation, TGF beta 1 and p27(kip1) antisense cDNA had a synergistic or additive inhibitory effect on cdk2 but not cyclin E-dependent kinase activity. Our data suggest that (1) TGF beta 1-mediated growth inhibition is accomplished through multiple pathways and (2) p27(kip1) has opposing effects on cdk2 and cyclin E activity in response to TGF beta 1.

Signaling pathway for nitric oxide generation with simulated ischemia in flow-adapted endothelial cells.

Ischemia in the intact ventilated lung (oxygenated ischemia) leads to endothelial generation of reactive oxygen species (ROS) and nitric oxide (NO). This study investigated the signaling pathway for NO generation with oxygenated ischemia in bovine pulmonary artery endothelial cells (BPAEC) that were flow adapted in vitro. BPAECs were cultured in an artificial capillary system and subjected to abrupt cessation of flow (ischemia) under conditions where cellular oxygenation was maintained. Immunoblotting and dichlorofluorescein/triazolofluorescein fluorescence were used to assess extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation and ROS/NO generation, respectively. ERK1/2 phosphorylation significantly increased during ischemia, whereas total ERK1/2 did not change. ERK1/2 phosphorylation was suppressed by an inhibitor of tyrosine phosphorylation (genestein), cholesterol-binding reagents (filipin or cyclodextrin), or inhibitors of ROS (diphenyleneiodonium, N-acetylcysteine, or catalase), suggesting a role for both membrane cholesterol and ROS in ERK1/2 activation. Ischemia resulted in a 1.8-fold increase in NO generation that was suppressed by inhibitors of ERK1/2 activation (PD-98059 or U-0126). A calmodulin inhibitor (calmidizolium) or removal of Ca2+ from the medium also blocked NO generation, indicating that endothelial NO synthase (eNOS) is the activated isoform. These results indicate ischemia induces NO generation (possibly through a membrane cholesterol-sensitive flow sensor), the ERK1/2 cascade mediates signaling from the sensor to eNOS, and ROS are required for ERK activation.

An immediate endothelial cell signaling response to lung ischemia.

Abrupt cessation of lung perfusion induces a rapid endothelial response that is not associated with anoxia but reflects loss of normal shear stress. This response includes membrane depolarization, H(2)O(2) generation, and increased intracellular Ca(2+). We evaluated these parameters immediately upon nonhypoxic ischemia using fluorescence videomicroscopy to image in situ endothelial cells in isolated, ventilated rat lungs. Lungs labeled with 4-(2-[6-(dioctylamino)-2-naphthalenyl]ethenyl)1-(3-sulfopropyl)-pyridinium (di-8-ANEPPS; a membrane potential probe), Amplex Red (an extracellular H(2)O(2) probe), or fluo 3-AM (a Ca(2+) indicator) were subjected to control perfusion followed by global ischemia. Endothelial di-8-ANEPPS fluorescence increased significantly within the first second of ischemia and stabilized at 15 s, indicating membrane depolarization by approximately 17 mV; depolarization was blocked by preperfusion with the K(+) channel agonist lemakalim. Increased H(2)O(2), inhibitable by catalase, was detected in the vascular space at 1-2 s after the onset of ischemia. Increased intracellular Ca(2+) was detected 10-15 s after the onset of ischemia; the initial increase was inhibited by preperfusion with thapsigargin. Thus the temporal sequence of the initial response of endothelial cells in situ to loss of shear stress (i.e., ischemia) is as follows: membrane depolarization, H(2)O(2) release, and increased intracellular Ca(2+).

Endothelial cellular response to altered shear stress.

Endothelial cells are normally exposed constantly to mechanical forces that significantly influence their phenotype. This symposium presented recent information concerning endothelial cell responses to shear stress associated with blood flow. Endothelial cell shear stress mechanosensors that have been proposed include membrane receptor kinases, integrins, G proteins, ion channels, intercellular junction proteins, membrane lipids (e.g., those associated with caveolae), and the cytoskeleton. These sensors are linked to signaling cascades that interact with or result in generation of reactive oxygen species, nitric oxide, and various transcription factors among other responses. Endothelial cells adapt to sustained shear stress, and either an increase or decrease from normal shear leads to signaling events. In vitro models for the study of endothelial cell responses must consider the pattern of shear stress (e.g., steady vs. oscillatory flow), the scaffold for cell growth (e.g., basement membrane or other cell types such as smooth muscle cells), and the extent of flow adaptation. These cellular responses have major relevance for understanding the pathophysiological effects of increased shear stress associated with hypertension or decreased shear stress associated with thrombotic occlusion.

Oxidative burst and NO generation as initial response to ischemia in flow-adapted endothelial cells.

Shear stress modulates endothelial physiology, yet the effect(s) of flow cessation is poorly understood. The initial metabolic responses of flow-adapted bovine pulmonary artery endothelial cells to the abrupt cessation of flow (simulated ischemia) was evaluated using a perfusion chamber designed for continuous spectroscopy. Plasma membrane potential, production of reactive O2 species (ROS), and intracellular Ca(2+) and nitric oxide (NO) levels were measured with fluorescent probes. Within 15 s after flow cessation, flow-adapted cells, but not cells cultured under static conditions, showed plasma membrane depolarization and an oxidative burst with generation of ROS that was inhibited by diphenyleneiodonium. EGTA-inhibitable elevation of intracellular Ca(2+) and NO were observed at approximately 30 and 60 s after flow cessation, respectively. NO generation was decreased in the presence of inhibitors of NO synthase and calmodulin. Thus flow-adapted endothelial cells sense the altered hemodynamics associated with flow cessation and respond by plasma membrane depolarization, activation of NADPH oxidase, Ca(2+) influx, and activation of Ca(2+)/calmodulin-dependent NO synthase. This signaling response is unrelated to cellular anoxia.

Lysosomal-type PLA2 and turnover of alveolar DPPC.

This study evaluated the role of a lysosomal-type phospholipase A2 (aiPLA(2)) in the degradation of internalized dipalmitoylphosphatidylcholine (DPPC) and in phospholipid synthesis by the rat lung. Uptake and degradation of DPPC were measured in isolated perfused rat lungs over 3 h following endotracheal instillation of [(3)H]DPPC in mixed unilamellar liposomes plus or minus MJ33, a specific inhibitor of lung aiPLA(2). Uptake of DPPC was calculated from total tissue-associated radiolabel, and degradation was calculated from the sum of radiolabel in degradation products. Both uptake and degradation were markedly stimulated by addition of 8-bromo-cAMP to the perfusate. MJ33 had no effect on DPPC uptake but decreased DPPC degradation at 3 h by approximately 40-50%. The effect of MJ33 on lung synthesis of DPPC was evaluated with intact rats over a 12- to 24-h period following intravenous injection of radiolabeled palmitate and choline. MJ33 treatment decreased palmitate incorporation into disaturated phosphatidylcholine of lamellar bodies and surfactant by approximately 65% at 24 h but had no effect on choline incorporation. This result is compatible with inhibition of the deacylation/reacylation pathway for DPPC synthesis. These results obtained with intact rat lungs indicate that aiPLA(2) is a major enzyme for degradation of internalized DPPC and also has an important role in DPPC synthesis.

Ca2+- and phosphatidylinositol 3-kinase-dependent nitric oxide generation in lung endothelial cells in situ with ischemia.

Endothelial cells generate nitric oxide (NO) in response to agonist stimulation or increased shear stress. In this study, we evaluated the effects of abrupt cessation of shear stress on pulmonary endothelial NO generation and its relationship to changes in intracellular Ca(2+). In situ endothelial generation of NO and changes in intracellular Ca(2+) in isolated, intact rat lungs were evaluated using fluorescence microscopy with diaminofluorescein diacetate, an NO probe, and Fluo-3, a Ca(2+) probe. The onset of increased NO generation in endothelial cells of subpleural microvessels in situ occurred between 30 and 90 s after onset of ischemia and was preceded by an increase in intracellular Ca(2+) due to both influx of extracellular Ca(2+) and release from intracellular stores. Flow cessation-induced NO generation in endothelial cells in situ was Ca(2+)-, calmodulin-, and PI3-kinase-dependent. The similarity of endothelial cell response (increased NO generation) to either increased flow or cessation of flow suggests that cells respond to an imposed alteration from a state of adaptation. This response to flow cessation may constitute a compensatory vasodilatatory mechanism and may play a role in signaling for cell proliferation and vascular remodeling.

Surface-expressed lamellar body membrane is recycled to lamellar bodies.

Monoclonal antibody (MAb) 3C9, an antibody generated to the lamellar body of rat lung type II pneumocytes, specifically labels the luminal face of the lamellar body membrane. To follow the retrieval of lamellar body membrane from the cell surface in these cells, MAb 3C9 was instilled into rat lungs. In vivo, it was endocytosed by type II cells but not by other lung cells. In type II cells that were isolated from rat lungs by elastase digestion and cultured on plastic for 24 h, MAb 3C9 first bound to the cell surface, then was found in endosomes, vesicular structures, and multivesicular bodies and, finally, clustered on the luminal face of lamellar body membranes. The amount internalized reached a plateau after 1.5 h of incubation and was stimulated with the secretagogue ATP. In double-labeling experiments, internalized MAb 3C9 did not completely colocalize with NBD-PC liposomes or the nonspecific endocytic marker TMA-DPH, suggesting that lamellar body membrane is retrieved back to existing lamellar bodies by a pathway different from that of bulk membrane and may be one pathway for surfactant endocytosis. The lamellar body membrane components are retrieved as subunits that are redistributed among the preexisting lamellar bodies in the cell.

Lung redox homeostasis: emerging concepts.

This symposium was organized to present some aspects of current research pertaining to lung redox function. Focuses of the symposium were on roles of pulmonary endothelial NADPH oxidase, xanthine oxidase (XO)/xanthine dehydrogenase (XDH), heme oxygenase (HO), transplasma membrane electron transport (TPMET), and the zinc binding protein metallothionein (MT) in the propagation and/or protection of the lung or other organs from oxidative injury. The presentations were chosen to reflect the roles of both intracellular (metallothionein, XO/XDH, and HO) and plasma membrane (NADPH oxidase, XO/XDH, and unidentified TPMET) redox proteins in these processes. Although the lung endothelium was the predominant cell type under consideration, at least some of the proposed mechanisms operate in or affect other cell types and organs as well.

1-Cys peroxiredoxin, a bifunctional enzyme with glutathione peroxidase and phospholipase A2 activities.

This report provides definitive evidence that the protein 1-Cys peroxiredoxin is a bifunctional ("moonlighting") enzyme with two distinct active sites. We have previously shown that human, rat, and bovine lungs contain an acidic Ca(2+)-independent phospholipase A(2) (aiPLA(2)). The cDNA encoding aiPLA(2) was found to be identical to that of a non-selenium glutathione peroxidase (NSGPx). Protein expressed using a previously reported E. coli construct which has a His-tag and 50 additional amino acids at the NH(2) terminus, did not exhibit aiPLA(2) activity. A new construct which contains the His-tag plus two extra amino acids at the COOH terminus when expressed in Escherichia coli generated a protein that hydrolyzed the sn-2 acyl chain of phospholipids at pH 4, and exhibited NSGPx activity with H(2)O(2) at pH 8. The expressed 1-Cys peroxiredoxin has identical functional properties to the native lung enzyme: aiPLA(2) activity is inhibited by the serine protease inhibitor, diethyl p-nitrophenyl phosphate, by the tetrahedral mimic 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33), and by 1-Cys peroxiredoxin monoclonal antibody (mAb) 8H11 but these agents have no effect on NSGPx activity; NSGPx activity is inhibited by mercaptosuccinate and by 1-Cys peroxiredoxin mAb 8B3 antibody which have no effect on aiPLA(2) activity. Mutation of Ser(32) to Ala abolishes aiPLA(2) activity, yet the NSGPx activity remains unaffected; a Cys(47) to Ser mutant is devoid of peroxidase activity but aiPLA(2) activity remains intact. These results suggest that Ser(32) in the GDSWG consensus sequence provides the catalytic nucleophile for the hydrolase activity of aiPLA(2), while Cys(47) in the PVCTTE consensus sequence is at the active site for peroxidase activity. The bifunctional catalytic properties of 1-Cys peroxiredoxin are compatible with a simultaneous role for the protein in the regulation of phospholipid turnover as well as in protection against oxidative injury.

Prolonged activation of the mitogen-activated protein kinase pathway is required for macrophage-like differentiation of a human myeloid leukemic cell line.

The role of the mitogen-activated protein kinase (MAPK) signal transduction pathway in the proliferation of mammalian cells has been well established. However, there are relatively few reports concerning cell differentiation being mediated by MAPK. The effect of phorbol 12-myristate 13-acetate (PMA) on cell differentiation and signal transduction in a human myeloid leukemia cell line, TF-1a, was investigated. When TF-1a cells were treated with 10(-6), 10(-7), 10(-8), and 10(-9) M PMA for 24 h, they underwent 98, 93, 91, and 51% macrophage-like differentiation, respectively. PMA treatment rapidly (10 min) induced phosphorylation of MAPK kinase (MEK and p44/42 MAPK), which persisted for at least 24 h. p44/42 MAPK immunoprecipitates from lysates of PMA-treated cells had increased ability to phosphorylate the transcription factor Elk-1. This is important because phosphorylated Elk-1 can be considered an "end-product" of the MAPK pathway. In contrast, treatment of TF-1a cells with granulocyte/macrophage-colony stimulating factor induced only transient activation of MEK and p44/42 MAPK (10-20 min) and an increase (approximately 50%) in cell proliferation, without any change in cellular differentiation. These results suggest that macrophage-like differentiation may be dependent on prolonged activation of the MAPK pathway. Additional support for this conclusion was obtained from experiments showing that treatment of TF-1a cells with antisense oligonucleotides for MEK1 coding sequences prior to adding PMA inhibited macrophage-like differentiation. Furthermore, transient transfection with an inactive, dominant-negative MEK mutant also inhibited PMA-induced differentiation, whereas transient transfection with a plasmid coding for constitutively activated MEK led to macrophage-like differentiation in the absence of PMA.

Lamellar body membrane turnover is stimulated by secretagogues.

Lamellar bodies are specialized cellular organelles used for storage of surfactant by alveolar type II cells of the lung. We utilized monoclonal antibody (MAb) 3C9, which recognizes an integral lamellar body-limiting membrane protein of 180 kDa, to follow lamellar body trafficking. (125)I-labeled MAb 3C9 bound to the surface of type II cells and was internalized by the cells in a time- and concentration-dependent manner that was inhibitable by excess unlabeled antibody. The internalized antibody remained undegraded over a 4-h time period. The L2 rat lung cell line that does not have lamellar bodies did not bind iodinated 3C9. Exposure of type II cells to the secretagogues ATP, phorbol 12-myristate 13-acetate, and cAMP resulted in a 1.5- to 2-fold enhancement of binding and uptake of MAb 3C9. Calphostin C inhibited phorbol 12-myristate 13-acetate-stimulated phospholipid secretion and also reduced binding and uptake of MAb 3C9 by type II cells. Treatment of type II cells with phenylarsine oxide to obstruct clathrin-mediated endocytosis had no effect on the internalization of MAb 3C9 while markedly blocking the uptake of surfactant protein A and transferrin. An actin-mediated process was important for lamellar body membrane uptake because incubation with cytochalasin D partially inhibited MAb 3C9 incorporation by type II cells. These studies are compatible with enhanced lamellar body membrane turnover associated with surfactant secretion and indicate that this process can be monitored by the trafficking of the antigen reporter MAb 3C9.

Depolarization-associated iron release with abrupt reduction in pulmonary endothelial shear stress in situ.

This study evaluated the roles of endothelial cell membrane potential and reactive oxygen species (ROS) in the increase of tissue free iron during lung ischemia. Oxygenated ischemia was produced in the isolated rat lung by discontinuing perfusion while ventilation with O2 was maintained. We have shown previously that tissue oxygenation is maintained in this model of ischemia and that biochemical changes are the result of an abrupt reduction in endothelial shear stress. With 1 hr oxygenated ischemia, generation of ROS, evaluated by oxidation of dichlorodihydrofluorescein (H2DCF) to fluorescent dichlorofluorescein, increased 8.0-fold, lung thiobarbituric acid reactive substances (TBARS) increased 3.4-fold, and lung protein carbonyl content increased 2.4-fold. Lung tissue free iron, measured in the lung homogenate with a fluorescent desferrioxamine derivative, increased 4.0-fold during ischemia. Pretreatment of lungs with thapsigargin abolished the increase in free iron with ischemia indicating that this effect is dependent on Ca2+ release from intracellular stores. Perfusion of lungs with high (25 mM) K+ to depolarize the endothelium also led to a significant increase in tissue free iron. Pretreatment of lungs with 35 microM cromakalim, a K+-channel agonist, significantly inhibited both ischemia-induced tissue oxidant injury and the increase in free iron with ischemia or with high K+ perfusion. A similar increase in free iron was observed when lungs were ventilated with either O2 or N2 during the ischemic period or were pre-perfused with an inhibitor of ROS production (diphenyleneiodonium). These results indicate that ROS generation is not required for ischemia-mediated iron release. Thus, ROS generation and iron release with ischemia are independent although both are subsequent to endothelial cell membrane depolarization.

Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis.

Metastasis is a frequent complication of cancer, yet the process through which circulating tumor cells form distant colonies is poorly understood. We have been able to observe the steps in early hematogenous metastasis by epifluorescence microscopy of tumor cells expressing green fluorescent protein in subpleural microvessels in intact, perfused mouse and rat lungs. Metastatic tumor cells attached to the endothelia of pulmonary pre-capillary arterioles and capillaries. Extravasation of tumor cells was rare, and it seemed that the transmigrated cells were cleared quickly by the lung, leaving only the endothelium-attached cells as the seeds of secondary tumors. Early colonies were entirely within the blood vessels. Although most models of metastasis include an extravasation step early in the process, here we show that in the lung, metastasis is initiated by attachment of tumor cells to the vascular endothelium and that hematogenous metastasis originates from the proliferation of attached intravascular tumor cells rather than from extravasated ones. Intravascular metastasis formation would make early colonies especially vulnerable to intravascular drugs, and this possibility has potential for the prevention of tumor cell attachment to the endothelium.

Simulated ischemia in flow-adapted endothelial cells leads to generation of reactive oxygen species and cell signaling.

We have previously shown that increased reactive oxygen species (ROS) generation occurs with ischemia in the oxygenated lung and have hypothesized that mechanotransduction is the initiating event. In the present study, we developed an in vitro model of oxygenated ischemia by interrupting medium flow to flow-adapted bovine pulmonary artery endothelial cells in an artificial capillary system. Cellular oxygenation during the "ischemic" period was maintained by perfusing medium over the abluminal surface of porous capillaries. Cells were assessed for ROS generation, nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) binding activities, and DNA synthesis using dichlorofluorescein fluorescence by flow cytometry and spectrofluorometry, electrophoretic mobility shift assay of nuclear extracts with NF-kappaB-specific or AP-1-specific (32)P-labeled oligonucleotides, and (3)H-thymidine incorporation into DNA. Cells that were flow adapted for 2 to 7 days with 1 to 2 dyne/cm(2) shear stress exhibited a 1.6- to 1.9-fold increase in ROS generation during 1 hour of simulated ischemia compared with continuously perfused cells. This effect was abolished by diphenyleneiodonium chloride (DPI), indicating a role for a flavoprotein such as NADPH oxidase. The increase in ROS generation with ischemia was similar for cells from low and high passages. With ischemia, flow-adapted cells exhibited increases of 1.7-fold in nuclear NF-kappaB and 1.5-fold in nuclear AP-1; these changes were abolished by pretreatment with N-acetylcysteine or DPI. Ischemia for 24 hours resulted in a 1.8-fold increase of (3)H-thymidine incorporation into DNA and a significant increase of cells entering the cell cycle, as indicated by flow cytometry with propidium iodide. We conclude that flow-adapted endothelial cells generate ROS with ischemia that results in activation of NF-kappaB and AP-1 and an increase of DNA synthesis. This effect is not mediated by hypoxia, implicating a role for mechanotransduction in ischemia-mediated cell signaling.

TNF-alpha-induced growth suppression of CD34+ myeloid leukemic cell lines signals through TNF receptor type I and is associated with NF-kappa B activation.

Conflicting results have been reported regarding the effect of TNF-alpha on the growth of human primitive hemopoietic cells. In this study, we have examined the effect of TNF-alpha on the proliferation of several CD34+/CD38+ (KG-1, TF-1) and CD34+/CD38- (KG-1a, TF-1a) myeloid leukemic progenitor cell lines. Our data show that TNF-alpha markedly inhibits the growth of these cells in both liquid and soft agar cultures. Addition of GM-CSF or IL-3 does not prevent TNF-alpha-induced growth inhibition. Flow cytometry analyses of propidium iodide-stained cells demonstrated cell death of all four cell lines, as judged by the presence of cells with hypodiploid DNA content after exposure of cells to TNF-alpha for 4 days. Annexin V assays detected apoptosis in TF-1, but not in TF-1a, KG-1, and KG-1a cells in terms of translocation of phosphatidylserine shortly after TNF-alpha treatment. Neutralizing anti-TNF receptor type I (TNFR-I; p55) Ab almost completely reversed TNF-alpha-induced growth inhibition in both liquid and soft agar cultures, whereas anti-TNFR-II (p75) Ab had only a marginal effect. TNF-alpha rapidly induced marked activation of nuclear transcription factor NF-kappa B in all 4 cell lines. The majority of this effect was abolished by the type I receptor Ab, whereas the type II receptor neutralizing Ab had no effect. Our data also show that TNF-alpha is incapable of inducing activation of the mitogen-activated protein kinase pathway in these leukemic cell lines.