Jacob I. Sznajder

Hypoxia-induced endocytosis of Na,K-ATPase in alveolar epithelial cells is mediated by mitochondrial reactive oxygen species and PKC-ζ

During ascent to high altitude and pulmonary edema, the alveolar epithelial cells (AEC) are exposed to hypoxic conditions. Hypoxia inhibits alveolar fluid reabsorption and decreases Na,K-ATPase activity in AEC. We report here that exposure of AEC to hypoxia induced a time-dependent decrease of Na,K-ATPase activity and a parallel decrease in the number of Na,K-ATPase alpha(1) subunits at the basolateral membrane (BLM), without changing its total cell protein abundance. These effects were reversible upon reoxygenation and specific, because the plasma membrane protein GLUT1 did not decrease in response to hypoxia. Hypoxia caused an increase in mitochondrial reactive oxygen species (ROS) levels that was inhibited by antioxidants. Antioxidants prevented the hypoxia-mediated decrease in Na,K-ATPase activity and protein abundance at the BLM. Hypoxia-treated AEC deficient in mitochondrial DNA (rho(0) cells) did not have increased levels of ROS, nor was the Na,K-ATPase activity inhibited. Na,K-ATPase alpha(1) subunit was phosphorylated by PKC in hypoxia-treated AEC. In AEC treated with a PKC-zeta antagonist peptide or with the Na,K-ATPase alpha(1) subunit lacking the PKC phosphorylation site (Ser-18), hypoxia failed to decrease Na,K-ATPase abundance and function. Accordingly, we provide evidence that hypoxia decreases Na,K-ATPase activity in AEC by triggering its endocytosis through mitochondrial ROS and PKC-zeta-mediated phosphorylation of the Na,K-ATPase alpha(1) subunit.

Ambient particulate matter accelerates coagulation via an IL-6–dependent pathway

The mechanisms by which exposure to particulate matter increases the risk of cardiovascular events are not known. Recent human and animal data suggest that particulate matter may induce alterations in hemostatic factors. In this study we determined the mechanisms by which particulate matter might accelerate thrombosis. We found that mice treated with a dose of well characterized particulate matter of less than 10 microM in diameter exhibited a shortened bleeding time, decreased prothrombin and partial thromboplastin times (decreased plasma clotting times), increased levels of fibrinogen, and increased activity of factor II, VIII, and X. This prothrombotic tendency was associated with increased generation of intravascular thrombin, an acceleration of arterial thrombosis, and an increase in bronchoalveolar fluid concentration of the prothrombotic cytokine IL-6. Knockout mice lacking IL-6 were protected against particulate matter-induced intravascular thrombin formation and the acceleration of arterial thrombosis. Depletion of macrophages by the intratracheal administration of liposomal clodronate attenuated particulate matter-induced IL-6 production and the resultant prothrombotic tendency. Our findings suggest that exposure to particulate matter triggers IL-6 production by alveolar macrophages, resulting in reduced clotting times, intravascular thrombin formation, and accelerated arterial thrombosis. These results provide a potential mechanism linking ambient particulate matter exposure and thrombotic events.

Hypoxia-induced alveolar epithelial-mesenchymal transition requires mitochondrial ROS and hypoxia-inducible factor 1.

Patients with acute lung injury develop hypoxia, which may lead to lung dysfunction and aberrant tissue repair. Recent studies have suggested that epithelial-mesenchymal transition (EMT) contributes to pulmonary fibrosis. We sought to determine whether hypoxia induces EMT in alveolar epithelial cells (AEC). We found that hypoxia induced the expression of alpha-smooth muscle actin (alpha-SMA) and vimentin and decreased the expression of E-cadherin in transformed and primary human, rat, and mouse AEC, suggesting that hypoxia induces EMT in AEC. Both severe hypoxia and moderate hypoxia induced EMT. The reactive oxygen species (ROS) scavenger Euk-134 prevented hypoxia-induced EMT. Moreover, hypoxia-induced expression of alpha-SMA and vimentin was prevented in mitochondria-deficient rho(0) cells, which are incapable of ROS production during hypoxia. CoCl(2) and dimethyloxaloylglycine, two compounds that stabilize hypoxia-inducible factor (HIF)-alpha under normoxia, failed to induce alpha-SMA expression in AEC. Furthermore, overexpression of constitutively active HIF-1alpha did not induce alpha-SMA. However, loss of HIF-1alpha or HIF-2alpha abolished induction of alpha-SMA mRNA during hypoxia. Hypoxia increased the levels of transforming growth factor (TGF)-beta1, and preincubation of AEC with SB431542, an inhibitor of the TGF-beta1 type I receptor kinase, prevented the hypoxia-induced EMT, suggesting that the process was TGF-beta1 dependent. Furthermore, both ROS and HIF-alpha were necessary for hypoxia-induced TGF-beta1 upregulation. Accordingly, we have provided evidence that hypoxia induces EMT of AEC through mitochondrial ROS, HIF, and endogenous TGF-beta1 signaling.

Isoproterenol increases Na+-K+-ATPase activity by membrane insertion of α-subunits in lung alveolar cells

Catecholamines promote lung edema clearance via β-adrenergic-mediated stimulation of active Na+ transport across the alveolar epithelium. Because alveolar epithelial type II cell Na+-K+-ATPase contributes to vectorial Na+ flux, the present study was designed to investigate whether Na+-K+-ATPase undergoes acute changes in its catalytic activity in response to β-adrenergic-receptor stimulation. Na+-K+-ATPase activity increased threefold in cells incubated with 1 μM isoproterenol for 15 min, which also resulted in a fourfold increase in the cellular levels of cAMP. Forskolin (10 μM) also stimulated Na+-K+-ATPase activity as well as ouabain binding. The increase in Na+-K+-ATPase activity was abolished when cells were coincubated with a cAMP-dependent protein kinase inhibitor. This stimulation, however, was not due to protein kinase-dependent phosphorylation of the Na+-K+-ATPase α-subunit; rather, it was the result of an increased number of α-subunits recruited from the late endosomes into the plasma membrane. The recruitment of α-subunits to the plasma membrane was prevented by stabilizing the cortical actin cytoskeleton with phallacidin or by blocking anterograde transport with brefeldin A but was unaffected by coincubation with amiloride. In conclusion, isoproterenol increases Na+-K+-ATPase activity in alveolar type II epithelial cells by recruiting α-subunits into the plasma membrane from an intracellular compartment in an Na+-independent manner.

Prompting physicians to address a daily checklist and process of care and clinical outcomes: A single-site study

RATIONALE Checklists may reduce errors of omission for critically ill patients. OBJECTIVES To determine whether prompting to use a checklist improves process of care and clinical outcomes. METHODS We conducted a cohort study in the medical intensive care unit (MICU) of a tertiary care university hospital. Patients admitted to either of two independent MICU teams were included. Intervention team physicians were prompted to address six parameters from a daily rounding checklist if overlooked during morning work rounds. The second team (control) used the identical checklist without prompting. MEASUREMENTS AND MAIN RESULTS One hundred and forty prompted group patients were compared with 125 control and 1,283 preintervention patients. Compared with control, prompting increased median ventilator-free duration, decreased empirical antibiotic and central venous catheter duration, and increased rates of deep vein thrombosis and stress ulcer prophylaxis. Prompted group patients had lower risk-adjusted ICU mortality compared with the control group (odds ratio, 0.36; 95% confidence interval, 0.13-0.96; P = 0.041) and lower hospital mortality compared with the control group (10.0 vs. 20.8%; P = 0.014), which remained significant after risk adjustment (odds ratio, 0.34; 95% confidence interval, 0.15-0.76; P = 0.008). Observed-to-predicted ICU length of stay was lower in the prompted group compared with control (0.59 vs. 0.87; P = 0.02). Checklist availability alone did not improve mortality or length of stay compared with preintervention patients. CONCLUSIONS In this single-site, preliminary study, checklist-based prompting improved multiple processes of care, and may have improved mortality and length of stay, compared with a stand-alone checklist. The manner in which checklists are implemented is of great consequence in the care of critically ill patients.

Augmentation of lung liquid clearance via adenovirus-mediated transfer of a Na,K-ATPase beta1 subunit gene.

Previous studies have suggested that alveolar Na,K-ATPases play an important role in active Na+ transport and lung edema clearance. We reasoned that overexpression of Na,K-ATPase subunit genes could increase Na,K-ATPase function in lung epithelial cells and edema clearance in rat lungs. To test this hypothesis we produced replication deficient human type 5 adenoviruses containing cDNAs for the rat alpha1 and beta1 Na,K-ATPase subunits (adMRCMValpha1 and adMRCMVbeta1, respectively). As compared to controls, adMRCMVbeta1 increased beta1 subunit expression and Na,K-ATPase function by 2. 5-fold in alveolar type 2 epithelial cells and rat airway epithelial cell monolayers. No change in Na,K-ATPase function was noted after infection with adMRCMValpha1. Rat lungs infected with adMRCMVbeta1, but not adMRCMValpha1, had increased beta1 protein levels and lung liquid clearance 7 d after tracheal instillation. Alveolar epithelial permeability to Na+ and mannitol was mildly increased in animals infected with adMRCMVbeta1 and a similar Escherichia coli lacZ-expressing virus. Our data shows, for the first time, that transfer of the beta1 Na,K-ATPase subunit gene augments Na,K-ATPase function in epithelial cells and liquid clearance in rat lungs. Conceivably, overexpression of Na,K-ATPases could be used as a strategy to augment lung liquid clearance in patients with pulmonary edema.

Alveolar Type 1 Cells Express the α2 Na,K-ATPase, Which Contributes to Lung Liquid Clearance

Abstract— The alveolar epithelium is composed of alveolar type 1 (AT1) and alveolar type 2 (AT2) cells, which represent ≈95% and ≈5% of the alveolar surface area, respectively. Lung liquid clearance is driven by the osmotic gradient generated by the Na,K-ATPase. AT2 cells have been shown to express the &agr;1 Na,K-ATPase. We postulated that AT1 cells, because of their larger surface area, should be important in the regulation of active Na+ transport. By immunofluorescence and electron microscopy, we determined that AT1 cells express both the &agr;1 and &agr;2 Na,K-ATPase isoforms. In isolated, ouabain-perfused rat lungs, the &agr;2 Na,K-ATPase in AT1 cells mediated 60% of the basal lung liquid clearance. The &bgr;-adrenergic agonist isoproterenol increased lung liquid clearance by preferentially upregulating the &agr;2 Na,K-ATPase protein abundance in the plasma membrane and activity in alveolar epithelial cells (AECs). Rat AECs and human A549 cells were infected with an adenovirus containing the rat Na,K-ATPase &agr;2 gene (Ad&agr;2), which resulted in the overexpression of the &agr;2 Na,K-ATPase protein and caused a 2-fold increase in Na,K-ATPase activity. Spontaneously breathing rats were also infected with Ad&agr;2, which increased &agr;2 protein abundance and resulted in a ≈250% increase in lung liquid clearance. These studies provide the first evidence that &agr;2 Na,K-ATPase in AT1 cells contributes to most of the active Na+ transport and lung liquid clearance, which can be further increased by stimulation of the &bgr;-adrenergic receptor or by adenovirus-mediated overexpression of the &agr;2 Na,K-ATPase.

AMP-activated protein kinase regulates CO2-induced alveolar epithelial dysfunction in rats and human cells by promoting Na,K-ATPase endocytosis

Hypercapnia (elevated CO(2) levels) occurs as a consequence of poor alveolar ventilation and impairs alveolar fluid reabsorption (AFR) by promoting Na,K-ATPase endocytosis. We studied the mechanisms regulating CO(2)-induced Na,K-ATPase endocytosis in alveolar epithelial cells (AECs) and alveolar epithelial dysfunction in rats. Elevated CO(2) levels caused a rapid activation of AMP-activated protein kinase (AMPK) in AECs, a key regulator of metabolic homeostasis. Activation of AMPK was mediated by a CO(2)-triggered increase in intracellular Ca(2+) concentration and Ca(2+)/calmodulin-dependent kinase kinase-beta (CaMKK-beta). Chelating intracellular Ca(2+) or abrogating CaMKK-beta function by gene silencing or chemical inhibition prevented the CO(2)-induced AMPK activation in AECs. Activation of AMPK or overexpression of constitutively active AMPK was sufficient to activate PKC-zeta and promote Na,K-ATPase endocytosis. Inhibition or downregulation of AMPK via adenoviral delivery of dominant-negative AMPK-alpha(1) prevented CO(2)-induced Na,K-ATPase endocytosis. The hypercapnia effects were independent of intracellular ROS. Exposure of rats to hypercapnia for up to 7 days caused a sustained decrease in AFR. Pretreatment with a beta-adrenergic agonist, isoproterenol, or a cAMP analog ameliorated the hypercapnia-induced impairment of AFR. Accordingly, we provide evidence that elevated CO(2) levels are sensed by AECs and that AMPK mediates CO(2)-induced Na,K-ATPase endocytosis and alveolar epithelial dysfunction, which can be prevented with beta-adrenergic agonists and cAMP.

Keratin 8 Phosphorylation by Protein Kinase C δ Regulates Shear Stress-mediated Disassembly of Keratin Intermediate Filaments in Alveolar Epithelial Cells

Phosphorylation of keratin intermediate filaments (IF) is known to affect their assembly state and organization; however, little is known about the mechanisms regulating keratin phosphorylation. In this study, we demonstrate that shear stress, but not stretch, causes disassembly of keratin IF in lung alveolar epithelial cells (AEC) and that this disassembly is regulated by protein kinase C δ-mediated phosphorylation of keratin 8 (K8) Ser-73. Specifically, in AEC subjected to shear stress, keratin IF are disassembled, as reflected by their increased solubility. In contrast, AEC subjected to stretch showed no changes in the state of assembly of IF. Pretreatment with the protein kinase C (PKC) inhibitor, bisindolymaleimide, prevents the increase in solubility of either K8 or its assembly partner K18 in shear-stressed AEC. Phosphoserine-specific antibodies demonstrate that K8 Ser-73 is phosphorylated in a time-dependent manner in shear-stressed AEC. Furthermore, we showed that shear stress activates PKC δ and that the PKC δ peptide antagonist, δ V1-1, significantly attenuates the shear stress-induced increase in keratin phosphorylation and solubility. These data suggested that shear stress mediates the phosphorylation of serine residues in K8, leading to the disassembly of IF in alveolar epithelial cells. Importantly, these data provided clues regarding a molecular link between mechanically induced signal transduction and alterations in cytoskeletal IF.

Adenovirus-mediated transfer of an Na+/K+ -ATPase β1 subunit gene improves alveolar fluid clearance and survival in hyperoxic rats

Pulmonary edema is cleared via active Na(+) transport by alveolar epithelial Na(+)/K(+)-ATPases and Na(+) channels. Rats exposed to acute hyperoxia have a high mortality rate, decreased Na(+)/K(+)-ATPase function, and decreased alveolar fluid clearance (AFC). We hypothesized that Na(+)/K(+)-ATPase subunit gene overexpression could improve AFC in rats exposed to hyperoxia. We delivered 4 x 10(9) PFU of recombinant adenoviruses containing rat alpha(1) and beta(1) Na(+)/K(+)-ATPase subunit cDNAs (adalpha(1) and adbeta(1), respectively) to rat lungs 7 days prior to exposure to 100% O(2) for 64 hr. As compared with controls and ad alpha(1), AFC in the adbeta(1) rats was increased by >300%. Permeability for large solutes was less in the ad beta(1) than in the other hyperoxia groups. Glutathione oxidation, but not superoxide dismutase activity, was increased only in the adbeta(1) group. Survival through 14 days of hyperoxia was 100% in the adbeta(1) group but was not different from hyperoxic controls in animals given adalpha(1). Our data show that overexpression of a beta(1) Na(+)/K(+)-ATPase subunit augments AFC and improves survival in this model of acute lung injury via antioxidant-independent mechanisms. Conceivably, restoration of AFC via gene transfer of Na(+)/K(+)-ATPase subunit genes may prove useful for the treatment of acute lung injury and pulmonary edema.