FOXO1 regulates VEGFA expression and promotes angiogenesis in healing wounds.

Angiogenesis is a critical aspect of wound healing. We investigated the role of keratinocytes in promoting angiogenesis in mice with lineage-specific deletion of the transcription factor FOXO1. The results indicate that keratinocyte-specific deletion of Foxo1 reduces VEGFA expression in mucosal and skin wounds and leads to reduced endothelial cell proliferation, reduced angiogenesis, and impaired re-epithelialization and granulation tissue formation. In vitro FOXO1 was needed for VEGFA transcription and expression. In a porcine dermal wound-healing model that closely resembles healing in humans, local application of a FOXO1 inhibitor reduced angiogenesis. This is the first report that FOXO1 directly regulates VEGFA expression and that FOXO1 is needed for normal angiogenesis during wound healing. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Nitric-oxide synthase-2 linkage to focal adhesion kinase in neutrophils influences enzyme activity and ß2 integrin function.

This investigation was to elucidate the basis for augmentation of nitric-oxide synthesis in neutrophils exposed to hyperbaric oxygen. Hyperoxia increases synthesis of reactive species leading to S-nitrosylation of ß-actin, which causes temporary inhibition of ß(2) integrin adherence. Impaired ß(2) integrin function and actin S-nitrosylation do not occur in neutrophils from mice lacking type-2 nitric-oxide synthase (iNOS) or when incubated with 1400W, an iNOS inhibitor. Similarly, effects of hyperoxia were abrogated in cells depleted of focal adhesion kinase (FAK) by treatment with small inhibitory RNA and those exposed to a specific FAK inhibitor concurrent with hyperoxia. Nitric oxide production doubles within 10 min exposure to hyperoxia but declines to approximately half-maximum production over an additional 10 min. Elevated nitric oxide production did not occur after FAK depletion or inhibition, or when filamentous actin formation was inhibited by cytochalasin D. Intracellular content of iNOS triples over the course of a 45-min exposure to hyperoxia and iNOS dimers increase in a commensurate fashion. Confocal microscopy and immunoprecipitation demonstrated that co-localization/linkage of FAK, iNOS, and filamentous actin increased within 15 min exposure to hyperoxia but then decreased below the control level. Using isolated enzymes in ex vivo preparations an association between iNOS and filamentous actin mediated by FAK could be demonstrated and complex formation was impeded when actin was S-nitrosylated. We conclude that iNOS activity is increased by an FAK-mediated association with actin filaments but peak nitric oxide production is transient due to actin S-nitrosylation during exposure to hyperoxia.

Thioredoxin reductase linked to cytoskeleton by focal adhesion kinase reverses actin S-nitrosylation and restores neutrophil ß(2) integrin function.

The investigation goal was to identify mechanisms for reversal of actin S-nitrosylation in neutrophils after exposure to high oxygen partial pressures. Prior work has shown that hyperoxia causes S-nitrosylated actin (SNO-actin) formation, which mediates ß(2) integrin dysfunction, and these changes can be reversed by formylmethionylleucylphenylalanine or 8-bromo-cyclic GMP. Herein we show that thioredoxin reductase (TrxR) is responsible for actin denitrosylation. Approximately 80% of cellular TrxR is localized to the cytosol, divided between the G-actin and short filamentous actin (sF-actin) fractions based on Triton solubility of cell lysates. TrxR linkage to sF-actin requires focal adhesion kinase (FAK) based on immunoprecipitation studies. S-Nitrosylation accelerates actin filament turnover (by mechanisms described previously (Thom, S. R., Bhopale, V. M., Yang, M., Bogush, M., Huang, S., and Milovanova, T. (2011) Neutrophil ß(2) integrin inhibition by enhanced interactions of vasodilator stimulated phosphoprotein with S-nitrosylated actin. J. Biol. Chem. 286, 32854-32865), which causes FAK to disassociate from sF-actin. TrxR subsequently dissociates from FAK, and the physical separation from actin impedes denitrosylation. If SNO-actin is photochemically reduced with UV light or if actin filament turnover is impeded by incubations with cytochalasin D, latrunculin B, 8-bromo-cGMP, or formylmethionylleucylphenylalanine, FAK and TrxR reassociate with sF-actin and cause SNO-actin removal. FAK-TrxR association can also be demonstrated using isolated enzymes in ex vivo preparations. Uniquely, the FAK kinase domain is the site of TrxR linkage. We conclude that through its scaffold function, FAK influences TrxR activity and actin S-nitrosylation.

Carbon monoxide inhalation increases microparticles causing vascular and CNS dysfunction.

We hypothesized that circulating microparticles (MPs) play a role in pro-inflammatory effects associated with carbon monoxide (CO) inhalation. Mice exposed for 1h to 100 ppm CO or more exhibit increases in circulating MPs derived from a variety of vascular cells as well as neutrophil activation. Tissue injury was quantified as 2000 kDa dextran leakage from vessels and as neutrophil sequestration in the brain and skeletal muscle; and central nervous system nerve dysfunction was documented as broadening of the neurohypophysial action potential (AP). Indices of injury occurred following exposures to 1000 ppm for 1h or to 1000 ppm for 40 min followed by 3000 ppm for 20 min. MPs were implicated in causing injuries because infusing the surfactant MP lytic agent, polyethylene glycol telomere B (PEGtB) abrogated elevations in MPs, vascular leak, neutrophil sequestration and AP prolongation. These manifestations of tissue injury also did not occur in mice lacking myeloperoxidase. Vascular leakage and AP prolongation were produced in naïve mice infused with MPs that had been obtained from CO poisoned mice, but this did not occur with MPs obtained from control mice. We conclude that CO poisoning triggers elevations of MPs that activate neutrophils which subsequently cause tissue injuries.

Neutrophil beta2 integrin inhibition by enhanced interactions of vasodilator-stimulated phosphoprotein with S-nitrosylated actin.

Production of reactive species in neutrophils exposed to hyperoxia causes S-nitrosylation of ß-actin, which increases formation of short actin filaments, leading to alterations in the cytoskeletal network that inhibit ß(2) integrin-dependent adherence (Thom, S. R., Bhopale, V. M., Mancini, D. J., and Milovanova, T. N. (2008) J. Biol. Chem. 283, 10822-10834). In this study, we found that vasodilator-stimulated protein (VASP) exhibits high affinity for S-nitrosylated short filamentous actin, which increases actin polymerization. VASP bundles Rac1, Rac2, cyclic AMP-dependent, and cyclic GMP-dependent protein kinases in close proximity to short actin filaments, and subsequent Rac activation increases actin free barbed end formation. Using specific chemical inhibitors or reducing cell concentrations of any of these proteins with small inhibitory RNA abrogates enhanced free barbed end formation, increased actin polymerization, and ß(2) integrin inhibition by hyperoxia. Alternatively, incubating neutrophils with formylmethionylleucylphenylalanine or 8-bromo-cyclic GMP activates either cyclic AMP-dependent or cyclic GMP-dependent protein kinase, respectively, outside of the short F-actin pool and phosphorylates VASP on serine 153. Phosphorylated VASP abrogates the augmented polymerization normally observed with S-nitrosylated actin, VASP binding to actin, elevated Rac activity, and elevated formation of actin free barbed ends, thus restoring normal ß(2) integrin function.

Vasculogenic stem cell mobilization and wound recruitment in diabetic patients: increased cell number and intracellular regulatory protein content associated with hyperbaric oxygen therapy.

Diabetic patients undergoing hyperbaric oxygen therapies (HBO(2)T) for refractory lower extremity neuropathic ulcers exhibit more than a twofold elevation (p=0.004) in circulating stem cells after treatments and the post-HBO(2)T CD34(+) cell population contains two- to threefold higher levels of hypoxia inducible factors-1, -2, and -3, as well as thioredoxin-1 (p<0.003), than cells present in blood before HBO(2)T. Skin margins obtained from 2-day-old abdominal wounds exhibit higher expression of CD133, CD34, hypoxia inducible factor-1, and Trx-1 vs. margins from refractory lower extremity wounds and expression of these proteins in all wounds is increased due to HBO(2)T (p<0.003). HBO(2)T is known to mobilize bone marrow stem cells by stimulating nitric oxide synthase. We found that nitric oxide synthase activity is acutely increased in patients' platelets following HBO(2)T and remains elevated for at least 20 hours. We conclude that HBO(2) T stimulates vasculogenic stem cell mobilization from bone marrow of diabetics and more cells are recruited to skin wounds.

FOXO1 expression in chondrocytes modulates cartilage production and removal in fracture healing.

Fracture healing is a multistage process characterized by inflammation, cartilage formation, bone deposition, and remodeling. Chondrocytes are important in producing cartilage that forms the initial anlagen for the hard callus needed to stabilize the fracture site. We examined the role of FOXO1 by selective ablation of FOXO1 in chondrocytes mediated by Col2α1 driven Cre recombinase. Experimental mice with lineage-specific FOXO1 deletion (Col2α1Cre+FOXO1L/L) and negative control littermates (Col2α1Cre-FOXO1L/L) were used for in vivo, closed fracture studies. Unexpectedly, we found that in the early phases of fracture healing, FOXO1 deletion significantly increased the amount of cartilage formed, whereas, in later periods, FOXO1 deletion led to a greater loss of cartilage. FOXO1 was functionally important as its deletion in chondrocytes led to diminished bone formation on day 22. Mechanistically, the early effects of FOXO1 deletion were linked to increased proliferation of chondrocytes through enhanced expression of cell cycle genes that promote proliferation and reduced expression of those that inhibit it and increased expression of cartilage matrix genes. At later time points experimental mice with FOXO1 deletion had greater loss of cartilage, enhanced formation of osteoclasts, increased IL-6 and reduced numbers of M2 macrophages. These results identify FOXO1 as a transcription factor that regulates chondrocyte behavior by limiting the early expansion of cartilage and preventing rapid cartilage loss at later phases.

Selective role for superoxide in InsP3 receptor-mediated mitochondrial dysfunction and endothelial apoptosis.

Reactive oxygen species (ROS) play a divergent role in both cell survival and cell death during ischemia/reperfusion (I/R) injury and associated inflammation. In this study, ROS generation by activated macrophages evoked an intracellular Ca2+ ([Ca2+]i) transient in endothelial cells that was ablated by a combination of superoxide dismutase and an anion channel blocker. [Ca2+]i store depletion, but not extracellular Ca2+ chelation, prevented [Ca2+]i elevation in response to O2*- that was inositol 1,4,5-trisphosphate (InsP3) dependent, and cells lacking the three InsP3 receptor (InsP3R) isoforms failed to display the [Ca2+]i transient. Importantly, the O2*--triggered Ca2+ mobilization preceded a loss in mitochondrial membrane potential that was independent of other oxidants and mitochondrially derived ROS. Activation of apoptosis occurred selectively in response to O2*- and could be prevented by [Ca2+]i buffering. This study provides evidence that O2*- facilitates an InsP3R-linked apoptotic cascade and may serve a critical function in I/R injury and inflammation.

Caveolae are an essential component of the pathway for endothelial cell signaling associated with abrupt reduction of shear stress.

Abrupt cessation of flow representing the acute loss of shear stress (simulated ischemia) to flow-adapted pulmonary microvascular endothelial cells (PMVEC) leads to reactive oxygen species (ROS) generation that signals for EC proliferation. We evaluated the role of caveolin-1 on this cellular response with mouse PMVEC that were preconditioned for 72 h to laminar flow at 5 dyn/cm(2) followed by stop of flow ("ischemia"). Preconditioning resulted in a 2.7-fold increase in cellular expression of K(ATP) (K(IR) 6.2) channels but no change in expression level of caveolin-1, gp91(phox), or MAP kinases. The initial response to ischemia in wild type cells was cell membrane depolarization that was abolished by gene targeting of K(IR) 6.2. The subsequent response was increased ROS production associated with activation of NADPH oxidase (NOX2) and then phosphorylation of MAP kinases (Erk, JNK). After 24 h of ischemia in wild type cells, the cell proliferation index increased 2.5 fold and the % of cells in S+G(2)/M phases increased 6-fold. This signaling cascade (cell membrane depolarization, ROS production, MAP kinase activation and cell proliferation) was abrogated in caveolin-1 null PMVEC or by treatment of wild type cells with filipin. These studies indicate that caveolin-1 functions as a shear sensor in flow-adapted EC resulting in ROS-mediated cell signaling and endothelial cell proliferation following the abrupt reduction in flow.

Identification of the amino acid sequence that targets peroxiredoxin 6 to lysosome-like structures of lung epithelial cells.

Peroxiredoxin 6 (Prdx6), an enzyme with glutathione peroxidase and PLA2 (aiPLA2) activities, is highly expressed in respiratory epithelium, where it participates in phospholipid turnover and antioxidant defense. Prdx6 has been localized by immunocytochemistry and subcellular fractionation to acidic organelles (lung lamellar bodies and lysosomes) and cytosol. On the basis of their pH optima, we have postulated that protein subcellular localization determines the balance between the two activities of Prdx6. Using green fluorescent protein-labeled protein expression in alveolar epithelial cell lines, we showed Prdx6 localization to organellar structures resembling lamellar bodies in mouse lung epithelial (MLE-12) cells and lysosomes in A549 cells. Localization within lamellar bodies/lysosomes was in the luminal compartment. Targeting to lysosome-like organelles was abolished by the deletion of amino acids 31-40 from the Prdx6 NH2-terminal region; deletion of the COOH-terminal region had no effect. A green fluorescent protein-labeled peptide containing only amino acids 31-40 showed lysosomal targeting that was abolished by mutation of S32 or G34 within the peptide. Studies with mutated protein indicated that lipid binding was not necessary for Prdx6 targeting. This peptide sequence has no homology to known organellar targeting motifs. These studies indicate that the localization of Prdx6 in acidic organelles and consequent PLA2 activity depend on a novel 10-aa peptide located at positions 31-40 of the protein.

Hyperbaric oxygen stimulates vasculogenic stem cell growth and differentiation in vivo.

We hypothesized that oxidative stress from hyperbaric oxygen (HBO(2), 2.8 ATA for 90 min daily) exerts a trophic effect on vasculogenic stem cells. In a mouse model, circulating stem/progenitor cell (SPC) recruitment and differentiation in subcutaneous Matrigel were stimulated by HBO(2) and by a physiological oxidative stressor, lactate. In combination, HBO(2) and lactate had additive effects. Vascular channels lined by CD34(+) SPCs were identified. HBO(2) and lactate accelerated channel development, cell differentiation based on surface marker expression, and cell cycle entry. CD34(+) SPCs exhibited increases in thioredoxin-1 (Trx1), Trx reductase, hypoxia-inducible factors (HIF)-1, -2, and -3, phosphorylated mitogen-activated protein kinases, vascular endothelial growth factor, and stromal cell-derived factor-1. Cell recruitment to Matrigel and protein synthesis responses were abrogated by N-acetyl cysteine, dithioerythritol, oxamate, apocynin, U-0126, neutralizing anti-vascular endothelial growth factor, or anti-stromal cell-derived factor-1 antibodies, and small inhibitory RNA to Trx reductase, lactate dehydrogenase, gp91(phox), HIF-1 or -2, and in mice conditionally null for HIF-1 in myeloid cells. By causing an oxidative stress, HBO(2) activates a physiological redox-active autocrine loop in SPCs that stimulates vasculogenesis. Thioredoxin system activation leads to elevations in HIF-1 and -2, followed by synthesis of HIF-dependent growth factors. HIF-3 has a negative impact on SPCs.

Mucosal Immunity and the FOXO1 Transcription Factors.

FOXO1 transcription factors affect a number of cell types that are important in the host response. Cell types whose functions are modulated by FOXO1 include keratinocytes in the skin and mucosal dermis, neutrophils and macrophages, dendritic cells, Tregs and B-cells. FOXO1 is activated by bacterial or cytokine stimulation. Its translocation to the nucleus and binding to promoter regions of genes that have FOXO response elements is stimulated by the MAP kinase pathway and inhibited by the PI3 kinase/AKT pathway. Downstream gene targets of FOXO1 include pro-inflammatory signaling molecules (TLR2, TLR4, IL-1ß, and TNF-α), wound healing factors (TGF-ß, VEGF, and CTGF) adhesion molecules (integrins-ß1, -ß3, -ß6, αvß3, CD11b, CD18, and ICAM-1), chemokine receptors (CCR7 and CXCR2), B cell regulators (APRIL and BLYS), T-regulatory modulators (Foxp3 and CTLA-4), antioxidants (GPX-2 and cytoglobin), and DNA repair enzymes (GADD45α). Each of the above cell types are found in oral mucosa and modulated by bacteria or an inflammatory microenvironment. FOXO1 contributes to the regulation of these cells, which collectively maintain and repair the epithelial barrier, formation and activation of Tregs that are needed to resolve inflammation, mobilization, infiltration, and activation of anti-bacterial defenses in neutrophils, and the homing of dendritic cells to lymph nodes to induce T-cell and B-cell responses. The goal of the manuscript is to review how the transcription factor, FOXO1, contributes to the activation and regulation of key leukocytes needed to maintain homeostasis and respond to bacterial challenge in oral mucosal tissues. Examples are given with an emphasis on lineage specific deletion of Foxo1 to explore the impact of FOXO1 on cell behavior, inflammation and susceptibility to infection.

FOXO1 Deletion Reverses the Effect of Diabetic-Induced Impaired Fracture Healing.

Type 1 diabetes impairs fracture healing. We tested the hypothesis that diabetes affects chondrocytes to impair fracture healing through a mechanism that involves the transcription factor FOXO1. Type 1 diabetes was induced by streptozotocin in mice with FOXO1 deletion in chondrocytes (Col2α1Cre+FOXO1L/L) or littermate controls (Col2α1Cre-FOXO1L/L) and closed femoral fractures induced. Diabetic mice had 77% less cartilage and 30% less bone than normoglycemics evaluated histologically and by micro-computed tomography. Both were reversed with lineage-specific FOXO1 ablation. Diabetic mice had a threefold increase in osteoclasts and a two- to threefold increase in RANKL mRNA or RANKL-expressing chondrocytes compared with normoglycemics. Both parameters were rescued by FOXO1 ablation in chondrocytes. Conditions present in diabetes, high glucose (HG), and increased advanced glycation end products (AGEs) stimulated FOXO1 association with the RANKL promoter in vitro, and overexpression of FOXO1 increased RANKL promoter activity in luciferase reporter assays. HG and AGE stimulated FOXO1 nuclear localization, which was reversed by insulin and inhibitors of TLR4, histone deacetylase, nitric oxide, and reactive oxygen species. The results indicate that chondrocytes play a prominent role in diabetes-impaired fracture healing and that high levels of glucose, AGEs, and tumor necrosis factor-α, which are elevated by diabetes, alter RANKL expression in chondrocytes via FOXO1.

Comparative analysis between CFSE flow cytometric and tritiated thymidine incorporation tests for beryllium sensitivity.

BACKGROUND: In this study, we evaluated alternative possibility for CFSE beryllium flow cytometric test against beryllium blood lymphocyte proliferation test (BeLPT) as a standard radioactive clinical screening method to identify sensitization to beryllium. METHODS: Delta PD (the ratio of divided cell population to the total number of cells with subtracted counts of unstimulated cells) of specific beryllium-induced pathogenic CD3+ CD4+ T-lymphocytes and stimulation index (SI) in CFSE proliferation test was compared with delta counts per minute (mean test CPM minus mean control CPM) and SI in radioactive blood BeLPT. RESULTS: Comparison analysis of CFSE and BeLPT demonstrated excellent agreement between delta PD and delta CPM (kappa = 0.845, P << 0.0001). We determined 6.8% positive subjects in the beryllium-exposed, Be-LPT-negative group. The decreased mean difference of these indexes to percentage of average and the long tail in the plot reflects increased sensitivity. CFSE/CD4+ T-cell proliferation assay has 100% specificity, significantly higher sensitivity and efficiency than BeLPT. CONCLUSIONS: Both delta PD, measured by the precursor frequencies method in CFSE assay and delta CPM, defined by tritiated thymidine in BeLPT, can be used for the enumeration of beryllium specific CD4+ T-cell proliferation and may substantially improve the quality of the early diagnosis of beryllium hypersensitivity.

Microparticles Impair Hypotensive Cerebrovasodilation and Cause Hippocampal Neuronal Cell Injury after Traumatic Brain Injury.

Endothelin-1 (ET-1), tissue plasminogen activator (tPA), and extracellular signal-regulated kinases-mitogen activated protein kinase (ERK-MAPK) are mediators of impaired cerebral hemodynamics after fluid percussion brain injury (FPI) in piglets. Microparticles (MPs) are released into the circulation from a variety of cells during stress, are pro-thrombotic and pro-inflammatory, and may be lysed with polyethylene glycol telomere B (PEG-TB). We hypothesized that MPs released after traumatic brain injury impair hypotensive cerebrovasodilation and that PEG-TB protects the vascular response via MP lysis, and we investigated the relationship between MPs, tPA, ET-1, and ERK-MAPK in that process. FPI was induced in piglets equipped with a closed cranial window. Animals received PEG-TB or saline (vehicle) 30-minutes post-injury. Serum and cerebrospinal fluid (CSF) were sampled and pial arteries were measured pre- and post-injury. MPs were quantified by flow cytometry. CSF samples were analyzed with enzyme-linked immunosorbent assay. MP levels, vasodilatory responses, and CSF signaling assays were similar in all animals prior to injury and treatment. After injury, MP levels were elevated in the serum of vehicle but not in PEG-TB-treated animals. Pial artery dilation in response to hypotension was impaired after injury but protected in PEG-TB-treated animals. After injury, CSF levels of tPA, ET-1, and ERK-MAPK were all elevated, but not in PEG-TB-treated animals. PEG-TB-treated animals also showed reduction in neuronal injury in CA1 and CA3 hippocampus, compared with control animals. These results show that serum MP levels are elevated after FPI and lead to impaired hypotensive cerebrovasodilation via over-expression of tPA, ET-1, and ERK-MAPK. Treatment with PEG-TB after injury reduces MP levels and protects hypotensive cerebrovasodilation and limits hippocampal neuronal cell injury.

Surfactant protein-A inhibits Aspergillus fumigatus-induced allergic T-cell responses.

BACKGROUND: The pulmonary surfactant protein (SP)-A has potent immunomodulatory activities but its role and regulation during allergic airway inflammation is unknown. METHODS: We studied changes in SP-A expression in the bronchoalveolar lavage (BAL) using a murine model of single Aspergillus fumigatus (Af) challenge of sensitized animals. RESULTS: SP-A protein levels in the BAL fluid showed a rapid, transient decline that reached the lowest values (25% of controls) 12 h after intranasal Af provocation of sensitized mice. Decrease of SP-A was associated with influx of inflammatory cells and increase of IL-4 and IL-5 mRNA and protein levels. Since levels of SP-A showed a significant negative correlation with these BAL cytokines (but not with IFN-gamma), we hypothesized that SP-A exerts an inhibitory effect on Th2-type immune responses. To study this hypothesis, we used an in vitro Af-rechallenge model. Af-induced lymphocyte proliferation of cells isolated from sensitized mice was inhibited in a dose-dependent manner by addition of purified human SP-A (0.1-10 microg/ml). Flow cytometric studies on Af-stimulated lymphocytes indicated that the numbers of CD4+ (but not CD8+) T cells were significantly increased in the parental population and decreased in the third and fourth generation in the presence of SP-A. Further, addition of SP-A to the tissue culture inhibited Af-induced IL-4 and IL-5 production suggesting that SP-A directly suppressed allergen-stimulated CD4+ T cell function. CONCLUSION: We speculate that a transient lack of this lung collectin following allergen exposure of the airways may significantly contribute to the development of a T-cell dependent allergic immune response.

Endothelial cell proliferation associated with abrupt reduction in shear stress is dependent on reactive oxygen species.

We have shown previously that flow-adapted endothelial cells respond to cessation of flow with cell membrane depolarization and increased production of reactive oxygen species, resulting in activation of transcription factors and increased DNA synthesis. This study utilized flow cytometry to evaluate cellular proliferation with ischemia and to determine the role of reactive oxygen species and apoptosis. PKH26-labeled rat pulmonary microvascular endothelial cells were seeded in an artificial capillary system and subjected to flow at 5 dynes/cm(2) for 96 h or for 72 h followed by 24 h of simulated "ischemia." Ischemia resulted in a 2.5-fold increase in the cellular proliferation index. Cell-cycle analysis showed G0/G1 arrest and decreased S plus G2/M during flow adaptation, whereas ischemia resulted in a three-fold increase of cells in S plus G2/M phases. Apoptotic cells as detected by TUNEL and annexin V binding assays were ~5% of total cells with no differences between static, flow-adapted, and "ischemic" groups. Reactive oxygen species production during a 1-h period following onset of ischemia was confirmed by oxidation of the fluorophore, dichlorofluorescein, and was inhibited by cromakalim, a K(ATP) channel agonist, or diphenyleneiodonium, a flavoprotein inhibitor. Cromakalim and diphenyleneiodonium also markedly inhibited cell proliferation in the flow-adapted ischemic cells, but had no effect on subconfluent cells cultured under static conditions. These results indicate reactive oxygen species-dependent endothelial cell proliferation in flow-adapted microvascular endothelial cells as a response to ischemia and indicate that this response is not a consequence of apoptosis.

Flow cytometric test for beryllium sensitivity.

BACKGROUND: Chronic beryllium disease (CBD) is an occupational granulomatous disorder characterized by hypersensitivity to beryllium, mediated by CD4+ T lymphocytes, and predominantly affects the lungs. In this disorder, lymphocyte proliferative responses to beryllium, measured by 3H thymidine incorporation, are used for diagnosis of CBD, for screening asymptomatic workers or former workers to detect unrecognized disease, and for surveillance as a bioassay to detect abnormal exposures. Problems with test variability and the use of radioactivity have recently led to the search for alternative methods. METHODS: We applied a 5,6-carboxyfluorescein diacetate succinimidyl ester flow cytometric technique for measurement of mitogen- and antigen-induced T-lymphocyte proliferation to a group of beryllium-exposed sensitized individuals and beryllium-unexposed controls. RESULTS: We detected mitogen and antigen proliferative responses in CD3+, CD4+, and CD8+ subpopulations. Phytohemagglutinin and Candida stimulated CD4+ and CD8+ T-cell responses, but beryllium appeared to stimulate only CD3+/CD4+ responses. CONCLUSIONS: This technique may provide a sensitive, nonradioactive alternative to the traditional proliferation tests that measure beryllium sensitivity. It offers the added specificity of enabling phenotypic description of the responding cell type and may prove to be easier to standardize for clinical use.

CD34+/CD45-dim stem cell mobilization by hyperbaric oxygen - changes with oxygen dosage.

Because hyperbaric oxygen treatment mobilizes bone marrow derived-stem/progenitor cells by a free radical mediated mechanism, we hypothesized that there may be differences in mobilization efficiency based on exposure to different oxygen partial pressures. Blood from twenty consecutive patients was obtained before and after the 1st, 10th and 20th treatment at two clinical centers using protocols involving exposures to oxygen at either 2.0 or 2.5 atmospheres absolute (ATA). Post-treatment values of CD34+, CD45-dim leukocytes were always 2-fold greater than the pre-treatment values for both protocols. Values for those treated at 2.5 ATA were significantly greater than those treated at 2.0 ATA by factors of 1.9 to 3-fold after the 10th and before and after the 20th treatments. Intracellular content of hypoxia inducible factors -1, -2, and -3, thioredoxin-1 and poly-ADP-ribose polymerase assessed in permeabilized CD34+ cells with fluorophore-conjugated antibodies were twice as high in all post- versus pre-treatment samples with no significant differences between 2.0 and 2.5 ATA protocols. We conclude that putative progenitor cell mobilization is higher with 2.5 versus 2.0 ATA treatments, and all newly mobilized cells exhibit higher concentrations of an array of regulatory proteins.