Elena M. Sorokina

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.

Lactate Stimulates Vasculogenic Stem Cells via the Thioredoxin System and Engages an Autocrine Activation Loop Involving Hypoxia-Inducible Factor 1

ABSTRACT The recruitment and differentiation of circulating stem/progenitor cells (SPCs) in subcutaneous Matrigel in mice was assessed. There were over one million CD34+ SPCs per Matrigel plug 18 h after Matrigel implantation, and including a polymer to elevate the lactate concentration increased the number of SPCs by 3.6-fold. Intricate CD34+ cell-lined channels were linked to the systemic circulation, and lactate accelerated cell differentiation as evaluated based on surface marker expression and cell cycle entry. CD34+ SPCs from lactate-supplemented Matrigel exhibited significantly higher concentrations of thioredoxin 1 (Trx1) and hypoxia-inducible factor 1 (HIF-1) than cells from unsupplemented Matrigel, whereas Trx1 and HIF-1 in CD45+ leukocytes were not elevated by lactate. Results obtained using small inhibitory RNA (siRNA) specific to HIF-1 and mice with conditionally HIF-1 null myeloid cells indicated that SPC recruitment and lactate-mediated effects were dependent on HIF-1. Cells from lactate-supplemented Matrigel had higher concentrations of phosphorylated extracellular signal-regulated kinases 1 and 2, Trx1, Trx reductase (TrxR), vascular endothelial growth factor (VEGF), and stromal cell-derived factor 1 (SDF-1) than cells from unsupplemented Matrigel. SPC recruitment and protein changes were inhibited by siRNA specific to lactate dehydrogenase, TrxR, or HIF-1 and by oxamate, apocynin, U0126, N-acetylcysteine, dithioerythritol, and antibodies to VEGF or SDF-1. Oxidative stress from lactate metabolism by SPCs accelerated further SPC recruitment and differentiation through Trx1-mediated elevations in HIF-1 levels and the subsequent synthesis of HIF-1-dependent growth factors.

Membrane depolarization is the trigger for PI3K/Akt activation and leads to the generation of ROS

Loss of fluid shear stress (ischemia) to the lung endothelium causes endothelial plasma membrane depolarization via ATP-sensitive K(+) (K(ATP)) channel closure, initiating a signaling cascade that leads to NADPH oxidase (NOX2) activation and ROS production. Since wortmannin treatment significantly reduces ROS production with ischemia, we investigated the role of phosphoinositide 3-kinase (PI3K) in shear-associated signaling. Pulmonary microvascular endothelial cells in perfused lungs subjected to abrupt stop of flow showed membrane depolarization and ROS generation. Stop of flow in flow-adapted mouse pulmonary microvascular endothelial cells in vitro resulted in the activation of PI3K and Akt as well as ROS generation. ROS generation in the lungs in situ was almost abolished by the PI3K inhibitor wortmannin and the PKC inhibitor H7. The combination of the two (wortmannin and H7) did not have a greater effect. Activation of NOX2 was greatly diminished by wortmannin, knockout of Akt1, or dominant negative PI3K, whereas membrane depolarization was unaffected. Ischemia-induced Akt activation (phosphorylation) was not observed with K(ATP) channel-null cells, which showed minimal changes in membrane potential with ischemia. Activation of Akt was similar to wild-type cells in NOX2-null cells, which do not generate ROS with ischemia. Cromakalim, a K(ATP) channel agonist, prevented both membrane depolarization and Akt phosphorylation with ischemia. Thus, Akt1 phosphorylation follows cell membrane depolarization and precedes the activation of NOX2. These results indicate that PI3K/Akt and PKC serve as mediators between endothelial cell membrane depolarization and NOX2 assembly.

Intracellular targeting of peroxiredoxin 6 to lysosomal organelles requires MAPK activity and binding to 14-3-3ε

Peroxiredoxin 6 (Prdx6), a bifunctional protein with GSH peroxidase and lysosomal-type phospholipase A(2) activities, has been localized to both cytosolic and acidic compartments (lamellar bodies and lysosomes) in lung alveolar epithelium. We postulate that Prdx6 subcellular localization affects the balance between the two activities. Immunostaining localized Prdx6 to lysosome-related organelles in the MLE12 and A549 alveolar epithelial cell lines. Inhibition of trafficking by brefeldin A indicated processing of the protein through the vesicular pathway. Trafficking of Prdx6 was decreased by inhibitors of PKC, ERK, and p38 MAPK. Immunocytochemistry, immunoprecipitation, and an in situ proximity ligation assay (Duolink) showed that binding of the lysosomal targeting sequence of Prdx6 (amino acids 31-40) to 14-3-3ε was dependent on activity of PKC, ERK, and p38 MAPK. Knockdown of 14-3-3ε with siRNA inhibited the lysosomal targeting of Prdx6. In vitro study with recombinant proteins by pull-down assay and surface plasmon resonance confirmed the interaction of Prdx6 and 14-3-3ε. These findings suggest that ERK and p38 MAPK regulate subcellular localization of Prdx6 by activation of 14-3-3ε as a chaperone protein, resulting in its translocation to acidic organelles.

PECAM-1 and caveolae form the mechanosensing complex necessary for NOX2 activation and angiogenic signaling with stopped flow in pulmonary endothelium

We showed that stop of flow triggers a mechanosignaling cascade that leads to the generation of reactive oxygen species (ROS); however, a mechanosensor coupled to the cytoskeleton that could potentially transduce flow stimulus has not been identified. We showed a role for KATP channel, caveolae (caveolin-1), and NADPH oxidase 2 (NOX2) in ROS production with stop of flow. Based on reports of a mechanosensory complex that includes platelet endothelial cell adhesion molecule-1 (PECAM-1) and initiates signaling with mechanical force, we hypothesized that PECAM-1 could serve as a mechanosensor in sensing disruption of flow. Using lungs in situ, we observed that ROS production with stop of flow was significantly reduced in PECAM-1(-/-) lungs compared with lungs from wild-type (WT) mice. Lack of PECAM-1 did not affect NOX2 activation machinery or the caveolin-1 expression or caveolae number in the pulmonary endothelium. Stop of flow in vitro triggered an increase in angiogenic potential of WT pulmonary microvascular endothelial cells (PMVEC) but not of PECAM-1(-/-) PMVEC. Obstruction of flow in lungs in vivo showed that the neutrophil infiltration as observed in WT mice was significantly lowered in PECAM-1(-/-) mice. With stop of flow, WT lungs showed higher expression of the angiogenic marker VEGF compared with untreated (sham) and PECAM-1(-/-) lungs. Thus PECAM-1 (and caveolae) are parts of the mechanosensing machinery that generates superoxide with loss of shear; the resultant ROS potentially drives neutrophil influx and acts as an angiogenic signal.

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.

Critical role of peroxiredoxin 6 in the repair of peroxidized cell membranes following oxidative stress.

Phospholipids are a major structural component of all cell membranes; their peroxidation represents a severe threat to cellular integrity and their repair is important to prevent cell death. Peroxiredoxin 6 (Prdx6), a protein with both GSH peroxidase and phospholipase A(2) (PLA(2)) activity, plays a critical role in antioxidant defense of the lung and other organs. We investigated the role of Prdx6 in the repair of peroxidized cell membranes in pulmonary microvascular endothelial cells (PMVEC) and isolated mouse lungs treated with tert-butyl hydroperoxide and lungs from mice exposed to hyperoxia (100% O(2)). Lipid peroxidation was evaluated by measurement of thiobarbituric acid reactive substances, oxidation of diphenyl-1-pyrenylphosphine, or ferrous xylenol orange assay. The exposure dose was varied to give a similar degree of lipid peroxidation at the end of exposure in the different models. Values for lipid peroxidation returned to control levels within 2 h after oxidant removal in wild-type PMVEC and perfused lungs but were unchanged in Pxdx6 null preparations. An intermediate degree of repair was observed with PMVEC and lungs that expressed only C47S or D140A mutant Prdx6; the former mutant does not have peroxidase activity, while the latter loses its PLA(2) activity. Prdx6 null mice showed markedly delayed recovery from lipid peroxidation during 20 h observation following exposure to hyperoxia. Thus, Prdx6 plays a critical role in the repair of peroxidized phospholipids in cell membranes and the recovery of lung cells from peroxidative stress; the peroxidase and PLA(2) activity each contribute to the recovery process.

A novel lysophosphatidylcholine acyl transferase activity is expressed by peroxiredoxin 6

The phospholipase A2 (PLA2) activity of peroxiredoxin (Prdx)6 has important physiological roles in the synthesis of lung surfactant and in the repair of peroxidized cell membranes. These functions require the activity of a lysophospholipid acyl transferase as a critical component of the phospholipid remodeling pathway. We now describe a lysophosphatidylcholine acyl transferase (LPCAT) activity for Prdx6 that showed a strong preference for lysophosphatidylcholine (LPC) as the head group and for palmitoyl CoA in the acylation reaction. The calculated kinetic constants for acylation were Km 18 μM and Vmax 30 nmol/min/mg protein; the Vmax was increased 25-fold by phosphorylation of the protein while Km was unchanged. Study of recombinant protein in vitro and in mouse pulmonary microvascular endothelial cells infected with a lentiviral vector construct indicated that amino acid D31 is crucial for LPCAT activity. A linear incorporation of labeled fatty acyl CoA into dipalmitoyl phosphatidylcholine (PC) indicated that LPC generated by Prdx6 PLA2 activity remained bound to the enzyme for the reacylation reaction. Prdx6 is the first LPCAT enzyme with demonstrated cytoplasmic localization. Thus, Prdx6 is a complete enzyme comprising both PLA2 and LPCAT activities for the remodeling pathway of PC synthesis or for repair of membrane lipid peroxidation.

Peroxiredoxin 6 homodimerization and heterodimerization with glutathione S-transferase pi are required for its peroxidase but not phospholipase A2 activity.

Peroxiredoxin 6 (Prdx6) is a unique 1-Cys member of the peroxiredoxin family with both GSH peroxidase and phospholipase A2 (PLA2) activities. It is highly expressed in the lung where it plays an important role in antioxidant defense and lung surfactant metabolism. Glutathionylation of Prdx6 mediated by its heterodimerization with GSH S-transferase π (πGST) is required for its peroxidatic catalytic cycle. Recombinant human Prdx6 crystallizes as a homodimer and sedimentation equilibrium analysis confirmed that this protein exists as a high affinity dimer in solution. Based on measurement of molecular mass, dimeric Prdx6 that was oxidized to the sulfenic acid formed a sulfenylamide during storage. After examination of the dimer interface in the crystal structure, we postulated that the hydrophobic amino acids L145 and L148 play an important role in homodimerization of Prdx6 as well as in its heterodimerization with πGST. Oxidation of Prdx6 also was required for its heterodimerization. Sedimentation equilibrium analysis and the Duolink proximity ligation assay following mutation of the L145 and L148 residues of Prdx6 to Glu indicated greatly decreased dimerization propensity reflecting the loss of hydrophobic interactions between the protein monomers. Peroxidase activity was markedly reduced by mutation at either of the Leu sites and was essentially abolished by the double mutation, while PLA2 activity was unaffected. Decreased peroxidase activity following mutation of the interfacial leucines presumably is mediated via impaired heterodimerization of Prdx6 with πGST that is required for reduction and re-activation of the oxidized enzyme.

Peroxiredoxin 6 phospholipid hydroperoxidase activity in the repair of peroxidized cell membranes

Although lipid peroxidation associated with oxidative stress can result in cellular death, sub-lethal lipid peroxidation can gradually resolve with return to the pre-exposure state. We have shown that resolution of lipid peroxidation is greatly delayed in lungs or cells that are null for peroxiredoxin 6 (Prdx6) and that both the phospholipase A2 and the GSH peroxidase activities of Prdx6 are required for a maximal rate of recovery. Like other peroxiredoxins, Prdx6 can reduce H2O2 and short chain hydroperoxides, but in addition can directly reduce phospholipid hydroperoxides. This study evaluated the relative role of these two different peroxidase activities of Prdx6 in the repair of peroxidized cell membranes. The His26 residue in Prdx6 is an important component of the binding site for phospholipids. Thus, we evaluated the lungs from H26A-Prdx6 expressing mice and generated H26A-Prdx6 expressing pulmonary microvascular endothelial cells (PMVEC) by lentiviral infection of Prdx6 null cells to compare with wild type in the repair of lipid peroxidation. Isolated lungs and PMVEC were exposed to tert-butyl hydroperoxide and mice were exposed to hyperoxia (> 95% O2). Assays for lipid peroxidation in wild type control and mutant lungs and cells showed ~4-fold increase at end-exposure. Control lungs and cells showed gradual resolution during a post-exposure recovery period. However, there was no recovery from lipid peroxidation by H26A-Prdx6 lungs or PMVEC. These studies confirm an important role for Prdx6 in recovery from membrane lipid peroxidation and indicate that reduction of H2O2 or short chain hydroperoxides does not play a role in the recovery process.