endothelial akt activation by hyperoxia: role in cell survival

Free Radical Biology & Medicine 40 (2006) 1108–1118www.elsevier.com/locate/freeradbiomed

Original Contribution

Endothelial Akt activation by hyperoxia: Role in cell survival

Aftab Ahmad a, Shama Ahmad a, Ling-Yi Chang b, Jerome Schaack c, Carl W. White a,⁎

aDepartment of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206, USAbDepartment of Medicine, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206, USA

cDepartment of Microbiology, School of Medicine, University of Colorado at Denver and Health Sciences Center, Aurora, CO 80045, USA

Received 14 March 2005; revised 14 October 2005; accepted 18 October 2005Available online 4 November 2005

Abstract

High oxygen concentrations (hyperoxia), often required in the treatment of preterm infants and critically ill patients, cause lung injury, targetingespecially the endothelium. Exposure of primary human lung microvascular endothelial cells (HLMVEC) to hyperoxia caused transient Aktactivation after 60 min, as determined by Western blot analysis of phosphorylated Ser 473 of Akt. Akt phosphorylation was also increased after 24h of hyperoxic exposure, which declined at 48 h. Adenoviral (Ad)-mediated expression of constitutively active myrAkt protected HLMVECagainst hyperoxic injury. Cell death due to hyperoxia (95% O2, 8 days), which was primarily necrotic, was substantial in control and Ad-LacZ-transduced cells, but was diminished by almost half in myrAkt-transduced cells. Hyperoxia caused increased cellular glucose consumption, aneffect that was amplified in cells transduced with myrAkt compared to the LacZ-transduced or the nontransduced controls. Increased glucoseconsumption in myrAkt-expressing cells was accompanied by increased phosphorylation of mTOR and p70 S6-kinase. Rapamycin treatmentdecreased glucose consumption in myrAkt-transduced cells to levels comparable to those in control and LacZ-transduced cells exposed tohyperoxia. Ultrastructural morphometric analyses demonstrated that mitochondria and endoplasmic reticulum were less swollen in myrAkt cellsrelative to controls exposed to hyperoxia. These studies demonstrate that early activation of Akt occurs in hyperoxia in HLMVEC. That this eventis a beneficial response is suggested by the finding that constitutive activation of Akt protects against hyperoxic stress, at least in part, bymaintaining mitochondrial integrity.© 2005 Elsevier Inc. All rights reserved.

Keywords: Hyperoxia; Akt; PKB; Endothelial; Glucose consumption; Necrosis; Mitochondria; Endoplasmic reticulum; Free radicals

Exposure to elevated concentrations of inspired oxygen(hyperoxia) often is required in the treatment of acuterespiratory failure of infants, children, and adults. Becauseprogressive lung injury due to hyperoxia occurs, such treatmentcan worsen the primary disease process and cause a poorerclinical outcome. In addition, hyperoxia can contribute todisease morbidity such as the chronic lung disease broncho-pulmonary dysplasia, which can occur in survivors of acuterespiratory failure in infants [1] and even adults [2]. The keyevents in the pathogenesis of, and defense against, hyperoxiclung injury are not fully understood. However, it is clear thatimpaired substrate utilization by mitochondria [3] and loss ofmitochondrial integrity [4,5] occur during hyperoxic treatment

⁎ Corresponding author.E-mail address: [email protected] (C.W. White).

0891-5849/$ - see front matter © 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.freeradbiomed.2005.10.045

and can contribute to impaired cell survival during hyperoxicstress. During exposure to hyperoxia, reactive oxygen species(ROS) production by mitochondria increases. This leads tomitochondrial damage resulting in impaired respiration, in partthrough inactivation of aconitase [6]. Hyperoxic adaptationcauses increased utilization of alternative substrates such asglucose and glutamine [7–9]. Thus, pathways that contribute toincreased utilization of these substrates also can protect againsthyperoxic injury [9,10]. The cell signaling mechanisms thatactivate, for example, glucose utilization during oxidative stressare not well understood.

Activation of the PI3-kinase/Akt pathway is associated withincreased cellular glucose metabolism as well as increasedsurvival under stress [11,12]. PI3-kinase activates Akt, which inturn phosphorylates a number of proteins involved in regulationof cell survival such as BAD, FOXO3a/FKHR-L1, CREB,IKK-kinase, glycogen synthase kinase-3 (GSK-3β), and

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1109A. Ahmad et al. / Free Radical Biology & Medicine 40 (2006) 1108–1118

MDM2. Akt inhibits apoptosis caused by growth factorwithdrawal [13,14] or hypoxia [15]. Growth factor withdrawalalso can cause a decline in glucose metabolism [16] involvingglucose transporters GLUT1 and GLUT4 [17]. Akt inhibitscytochrome c release and maintains integrity of mitochondriathrough upregulation of mitochondrial hexokinases [18,19].PKB/Akt can be transiently activated by exposure to hypoxia[20], and hypoxic preconditioning also can protect againstoxidative stresses such as hyperoxia [21] and ischemia–reperfusion [22]. The role of PI3k/Akt in cell survival is furtherunderscored by the finding that loss of PTEN, a tumorsuppressor gene, causes constitutive activation of the PI3-kinase/Akt pathway leading to diminished cell death [23].

Lu et al. [24] reported that targeted delivery of myrAkt tolung epithelium can diminish acute lung injury and death due tohyperoxia. In a related study in mice, keratinocyte growth factorprotected the epithelium against hyperoxic insult through anAkt-dependent pathway, but did not increase survival. Howev-er, the microvascular endothelium is a principal target of injury,both in rodents [25] and in primates [26,27], after lethal orsublethal hyperoxia exposure. Therefore, we sought todetermine the effect of hyperoxia on the activation of Akt inhuman lung microvascular endothelial cells, as well as the effectof transducing these cells with active Akt before hyperoxicstress. Importantly, we elucidated a novel signaling mechanismmediating critical metabolic changes required for Akt-inducedendothelial protection. Here we show that early Akt activationoccurs after exposure to hyperoxia and that this may be ofbenefit because sustained overexpression of constitutivelyactive Akt protects human lung microvascular endothelialcells against hyperoxia-induced death and loss of mitochondrialintegrity.

Materials and methods

Materials

Antibodies that recognize Akt, phospho-Akt (Ser 473), p70S6-kinase (p70S6K), phospho-p70 S6-kinase (Thr 389),mTOR, and phospho-mTOR (Ser 2448) were procured fromCell Signaling Technology, Inc. (Beverly, MA, USA). Primarylung microvascular endothelial cells (HLMVEC) and endothe-lial cell growth medium were obtained from Cambrex(Walkersville, MD, USA).

Cell culture and exposure

HLMVEC were cultured in endothelial cell basal medium(EBM-2) supplemented with VEGF, human FGF, human EGF,hydrocortisone, ascorbic acid, insulin-like growth factor-1, GA-1000 (gentamicin/amphotericin-B), and 5% fetal bovine serumas per the manufacturer's protocol.

HLMVECwere exposed to air (21% O2/5% CO2/95% N2) orhyperoxia (95% O2/5% CO2). Fresh medium was supplied dailyduring exposure. For survival and electron microscopic studiescells were exposed to air for 2 days or hyperoxia for 8 days inorder for cultures to be matched for confluence. Earlier studies

have shown that confluency of cells is an important factor inhyperoxia-induced toxicity, such that subconfluent cells showedgreater resistance to cell death in hyperoxia than confluent cells[28]. Exposures were carried out at sea level pressure (760 mmHg) except when exposed to hyperoxia for short intervals of upto 120 min for determining Akt phosphorylation (635 mm Hg).Hypoxic exposure was performed in 5% CO2/95% N2 [29]. Forexposures for which Akt phosphorylation was assessed,medium without serum and growth factors was used.

Cell viability assay

After exposure of cells to oxidant stress (8 days hyperoxia),cell death was assessed by using a Vybrant Apoptosis DetectionKit (Molecular Probes, Eugene, OR, USA) as describedpreviously [30]. Briefly, cells were trypsinized, pelleted, andresuspended in annexin binding buffer and incubated withAlexa Fluor annexin V and the fluorescent DNA-binding dyeSYTOX green for 15 min at room temperature in the darkaccording to the manufacturer's protocol. The samples werethen put on ice, and sample volume was brought to 0.5 ml.Analysis was carried out using a FACSCalibur flow cytometer(Becton–Dickinson, San Jose, CA, USA) with the FITC signaldetector FL1. Annexin-positive cells were determined asdescribed by the manufacturer. The population separates intothree groups: live cells with only a very low level offluorescence, apoptotic cells with moderate green fluorescence,and necrotic cells with high-intensity green fluorescence. Cellsexposed to hydrogen peroxide provided a positive control forapoptosis.

In order to assess nuclear morphology, HLMVEC weregrown in chamber slides and exposed to air or hyperoxia for8 days. Nuclear size was estimated by staining cells with 2 μg/ml DAPI for 10 min as described [31]. Images of nuclei werecaptured using an inverted Zeiss 200 M microscope with aSutter Instruments (Novarto, CA, USA) DG4 175-W xenonlamp and a Cooke Sensicam camera (Romulus, MI, USA).Nuclear area was then estimated using the Slidebook v4.1.0software (Intelligent Imaging Innovations, Inc., Denver, CO,USA).

2-[3H]Deoxyglucose uptake

2-Deoxyglucose uptake was measured as described previ-ously [32]. Briefly, cells were grown in endothelial cell growthmedium EBM-2 containing growth factors, in six-well plates.Cells were either not transduced or transduced with adenovirus(Ad) vectors encoding LacZ (Ad-LacZ) or myrAkt (Ad-myrAkt) at a multiplicity of 10 plaque-forming units (pfu)/cell. Twenty-four hours posttransduction, medium from cellswas changed to glucose-free DMEM and maintained for anadditional 24 h. For measuring 2-[3H]deoxyglucose uptake,cells were washed twice with Krebs Ringer–phosphate–Hepes(KRP-Hepes) buffer (25 mM Hepes, pH 7.5, 140 mM NaCl, 5mM KCl, 1 mM CaCl2, 1.2 mM KH2PO4, 2.5 mM MgSO4, 5mM NaHCO3, and 0.1% BSA) and incubated in 950 μl KRP-Hepes buffer for 60 min. Cells were then exposed to air (21%

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O2/5% CO2/95% N2) or hyperoxia (95% O2/5% CO2) for 60min. Glucose uptake was measured by adding 20 μl glucosemixture (5 mM 2-deoxyglucose, 0.5 μCi 2-[3H]deoxyglucose inKRP-Hepes buffer) to 980 μl KRP-Hepes buffer and incubatingfor 20 min at 37°C. Nonspecific glucose uptake was measuredin the presence of 10 μM cytochalasin B. Uptake wasterminated by washing the cells three times with 1 ml of ice-cold phosphate-buffered saline. Cells were subsequently lysedwith 0.5 ml of 0.5 M NaOH solution containing 0.1% SDS.Cell-associated radioactivity was measured by a liquidscintillation counter.

Glucose consumption assay

Glucose consumed by cells was assayed using a commer-cially available glucose assay (Trinder) kit from Sigma (St.Louis, MO, USA). After exposure, medium was collected andcentrifuged to remove cellular debris. One milliliter of the assayreagent was mixed with 50 μl of medium and incubated at 25°Cfor 18 min, after which the absorbance was recorded at 505 nmusing a Shimadzu UV-1601 UV–Vis spectrophotometer.Glucose concentration in medium supernatants was estimatedagainst a standard curve. Glucose consumed was estimated bysubtracting total glucose in cell medium from that in freshunexposed medium and plotted, with data expressed per numberof cells.

Akt kinase assay

Akt kinase activity was assayed using an Akt1/PKBαimmunoprecipitation kinase assay kit (Upstate Biotechnology(Lake Placid, NY, USA). The assay measures phosphotransfer-ase activity in an immunocomplex formed between the Akt1/PKBα pleckstrin homology domain antibody and active Akt/PKBα. Briefly, cells were harvested in 50 mM Tris–HCl, pH7.5, containing 50 mMNaF, 1% (v/v) 2-mercaptoethanol, 1 mMEDTA, 1 mM EGTA, 1% Triton X-100, 5 mM sodiumpyrophosphate, 10 mM sodium β-glycerophosphate, 1 mMNa3VO4, 0.1 mM PMSF, 1 μM microcystin, and 1 μg/ml eachleupeptin, aprotinin, and pepstatin. The precipitated Akt/PKBαwas used to transfer the γ-phosphate of [γ-32P]ATP to a specificsubstrate peptide corresponding to the sequence containing aphosphorylation site from GSK-3β. The peptide substrate wasthen separated using P81 phosphocellulose paper and washedand the amount of radioactivity quantitated using a scintillationcounter.

Electron microscopy

HLMVEC were cultured as described above and fixed insitu with 2.5% glutaraldehyde. Cells were scraped, pelleted,and prepared for electron microscopic examination followingstandard procedures. Three samples were cut for each culturecondition. The ultrathin sections were mounted on 100-meshgrids. The sections were systematically surveyed in a PhilipCM 10 electron microscope. The first 10 grids that containedan endothelial cell(s) at the upper left corner of the grid

space were photographed at a magnification of 100,000×.Volume densities of the mitochondria and endoplasmicreticulum were determined by point counting [33]. Volume-to-surface ratios of the mitochondrial cristae and ERcisternae were determined by measuring the profile areasand lengths of the organelles with image analysis software.Results are expressed as means ± standard error.

Western blotting

Cell extracts were prepared in lysis buffer containing 20 mMTris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA,1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin, and 1μM microcystin. Samples containing 40 μg of protein wereseparated on a 4–15% SDS–PAGE gel (Gradipore Ltd.,Australia) and transferred onto nitrocellulose membrane (PallGelman, Pensacola, FL, USA) in 25 mM Tris, 190 mM glycine,and 20% methanol, pH 8.3, at 90 V for 2 h using a transblotapparatus (Bio-Rad Laboratories, Hercules, CA, USA). Themembrane was blocked using 5% milk protein in TBScontaining 1% Tween 20 (TBS-T). Phosphorylated Akt wasdetected using a polyclonal anti-phospho-Ser 473 antibody(Cell Signaling Technology) followed by an HRP-conjugatedanti-rabbit polyclonal. The blots were developed usingenhanced chemiluminescence detection with a SupersignalPico Ultradetection Reagent (Pierce, Rockford, IL, USA). Forcontrols, blots were stripped with a stripping buffer (GenoTechnology, Inc., St. Louis, MO, USA) for 15 min, blockedwith 5% milk protein in TBS-T, and reprobed.

Adenovirus vectors

Adenoviruses were propagated in HEK 293 cells [34] andpurified using consecutive CsCl step and isopycnic gradientcentrifugation [35]. Viral titers were determined spectrophoto-metrically after dilution in water with 1 OD at 260 nm = 1012

viral particles/ml, and the particle:pfu ratio was approximately100:1. Cells were transduced with 10 pfu/cell of Ad-myrAkt (akind gift from Dr. Kenneth Walsh) [36] or Ad-LacZ [37].Hyperoxic exposure was started 24 h after transduction, withmedium changed every 24 h for 8 days.

Statistical analysis

All statistical analyses were performed with the JMPsoftware (SAS Institute, Cary, NC, USA). Data are representedas means ± SEM of n ≥ 3 independent experiments and werecompared by ANOVA followed by the Tukey–Kramer test formultiple comparisons. A p value of b0.05 was consideredsignificant.

Results

Changes in oxygen tension can cause rapid changes inprotein phosphorylation, leading to activation or inactivation ofdistinct signaling cascades. Previously, Akt phosphorylation

Fig. 1. Effects of hyperoxia on Akt phosphorylation in HLMVEC. HLMVEC were plated in 100-mm dishes and grown to 80% confluence. (A) Cells were serumstarved for 24 h before exposure to 95% O2/5% CO2 (Oxy) or 21% O2/5% CO2 (Air) for the indicated time periods. (B) Cells were exposed to air (21% O2/5% CO2),hyperoxia (95% O2/5% CO2), or hypoxia (0% O2/5% CO2) for 24 h in absence of serum. Blots were initially probed for phospho Akt (Ser 473), stripped, andsubsequently probed for Akt. (C) Cells were exposed to air (21% O2/5% CO2) or hyperoxia (95% O2/5% CO2) for the indicated time.

Fig. 2. Effects of adenoviral-mediated Akt transduction on cell death in HLMVEC exposed to hyperoxia. HLMVEC were transduced with Ad-LacZ and Ad-myrAkt.Twenty-four hours posttransduction, medium was changed and cells were exposed to air or hyperoxia. Additional medium changes were carried out every 24 h. (A)Cell death was assessed by flow cytometry using the Vybrant apoptosis detection kit. (B) Nuclear staining was carried out using DAPI. Mean relative areas of nuclearsize are plotted. *represents statistical difference (p b 0.05) from control in air, #represents statistical difference from control and LacZ-transduced cells exposed tohyperoxia. For experimental details, see Materials and methods.


Fig. 3. Effects of Akt transduction on glucose uptake by HLMVEC in hyperoxia.Untransduced, Ad-LacZ-transduced, and Ad-myrAkt-transduced HLMVEC(2 × 105 cells) were glucose starved for 24 h and then exposed to air or hyperoxiafor 60 min in Krebs Ringer buffer. Cells were subsequently treated with KrebsRinger buffer containing 3H-labeled 2-deoxyglucose and 5 mM glucose for 20min. Cells were washed and subsequently lysed. Radioactivity was measuredand plotted. *represents statistical difference (p b 0.05) from control in air,#represents statistical difference from control and LacZ-transduced cells exposedto hyperoxia. For experimental details, see Materials and methods.


was found to be enhanced by brief hypoxic exposure [20]. Ourprevious studies also showed increased PI3-kinase activity inHLMVEC after 24 h of hypoxia. To determine if hyperoxiacould modulate Akt activation, phosphorylation of Akt afterexposure to hyperoxia was evaluated. Initial increases in Aktphosphorylation (Ser 473) occurred maximally after 60 min ofexposure (Fig. 1A). The phosphorylated form of Akt remainedabove its basal level in air after 24 h of hyperoxia but declinedby 48 h (Figs. 1B and 1C). Total Akt was unchanged at 24h but decreased by 48 h of exposure. This was in sharpcontrast to cells exposed to hypoxia for 24 h, in which therewas a 6.8-fold increase in the phosphorylated relative to

Fig. 4. Effects of Akt transduction on glucose consumption by HLMVEC in hyperoxwere exposed to air or hyperoxia in the presence or absence of rapamycin (100 nM). Minhibitor was added, and medium were collected again at 48 h after an additional 24 hsummed and expressed per cell number. *Statistical difference (p b 0.05) from contrexposed to hyperoxia. For experimental details, see Materials and methods.

nonphosphorylated form (Fig. 1B), despite a decrease in totalAkt levels.

To ascertain whether Akt activation played a role inhyperoxic injury or defense of the microvasculature, we useda constitutively active myristylated form of Akt (myrAkt). TheAkt kinase activity of HLMVEC transduced with Ad-myrAktwas significantly greater than that of Ad-LacZ-transduced ornontransduced controls (data not shown). HLMVEC transducedwith Ad-LacZ or Ad-myrAkt or untransduced control cells wereexposed to 21% O2 (“air”) for 2 days or hyperoxia for 8 days inorder for cultures to be matched for confluence. In air, cellscontinued to grow at all times. In contrast, cells in hyperoxiashowed growth arrest within 24–48 h. In the present study,hyperoxia caused increased necrotic cell death, in bothuntransduced and Ad-LacZ-transduced controls (Fig. 2).Death due to hyperoxia of cells transduced by constitutivelyactive Akt, myrAkt, was decreased by almost 50% relative tothe two control groups. Because hyperoxic death can cause cellswelling, including an increase in nuclear size, we alsoevaluated nuclear morphology. In our studies, increased nucleararea was observed in the cells exposed to hyperoxia (Fig. 2B).However, nuclear area of myrAkt-transduced cells in hyperoxiawas decreased by almost half relative to the two controls.

Transient Akt activation also can cause cellular biochemicalchanges, most notably in glucose metabolism. We have shownthat hyperoxia increases 2-deoxyglucose uptake after 4 h ofexposure and that this uptake was inhibitable by rapamycin[32]. Therefore, we investigated 2-deoxyglucose uptake inhyperoxic exposure at a time when endogenous Akt phosphor-ylation is increased. Ad-myrAkt-transduced cells exhibitedincreased glucose uptake relative to nontransduced cells or cellstransduced with Ad-LacZ. However, in each of these threegroups, exposure to hyperoxia for this brief interval (1 h) did notcause elevation of glucose uptake relative to that observed in air(Fig. 3).

Oxidative stress also can cause cellular biochemical changesassociated with adaptation, injury, or death. With prolonged

ia. Untransduced, Ad-LacZ-transduced, and Ad-myrAkt-transduced HLMVECedium was collected after exposure for 24 h. Medium was changed at 24 h, freshhyperoxic exposure. Thus, glucose consumption from 0–24 h and 24–48 h wasol medium in air, #statistical difference from control and LacZ-transduced cells

Fig. 5. Effects of Akt transduction and hyperoxia on mTOR and p70 S6-kinase phosphorylation. Untransduced, Ad-LacZ-transduced, and Ad-myrAkt-transducedHLMVEC were exposed to air (21% O2/5% CO2) or hyperoxia (95% O2/5% CO2) for 48 h with medium change after the initial 24 h. Cells were lysed and 25 μgprotein was loaded per lane. Blots were initially probed for phospho-mTOR (Ser 2448), stripped, and subsequently probed with mTOR. A separate blot was alsoprobed for phospho-p70 S6-kinase (Thr 398), stripped, and subsequently probed with p70 S6-kinase. For experimental details, see Materials and methods.


exposure (48 h), hyperoxia causes increased glucose utilizationby cells. Therefore, we examined glucose utilization ofHLMVEC in the presence or absence of Akt transduction.Fig. 4 shows that there was an additional increase in glucoseutilization of cells transduced with myrAkt during 48 h ofhyperoxic exposure relative to nontransduced or LacZ-trans-duced controls. There was also a significant increase in glucoseutilization by Akt-transduced cells exposed to 21% O2 relativeto the two control groups. In the presence of rapamycin, whichinhibits mTOR activity, the glucose consumption of myrAkt-

Fig. 6. Effects of adenoviral-mediated transduction of Akt on cell morphology of HLMmyrAkt-transduced HLMVEC were exposed to air (2 days) or hyperoxia (8 days)microscopy. Representative images are displayed. For experimental details, see Mat

transduced cells was reduced to levels comparable to controlnontransduced or LacZ-transduced cells.

Assessment of downstream targets of Akt showed anincrease in phosphorylated mTOR and p70 S6-kinase proteins(Fig. 5).

In the process of causing cell injury and death, oxidativeinsults can cause important ultrastructural changes. Hyperoxialeads to alterations in mitochondrial morphology [4]. In order toassess the role of constitutively active Akt in limiting hyperoxicdamage, we examined cell structural changes in HLMVEC

VEC after exposure to hyperoxia. Untransduced, Ad-LacZ-transduced, and Ad-. After 8 days of hyperoxic exposure, cells were visualized by phase-contrasterials and methods.


exposed to either 21% O2 for 2 days or 95% O2 for 8 days atboth the light (Fig. 6) and the electron microscopic levels (Fig.7). A significant increase in detached or rounded and/or necroticcells was observed both in untreated and in LacZ-transducedcells compared to myrAkt-transduced cells exposed to hyper-oxia. There were no significant differences in the death of cellsexposed to air among control nontransduced, LacZ-transduced,or myrAkt-transduced cells. Fig. 7 shows electron micrographs

Fig. 7. Effects of Akt transduction and/or hyperoxia on ultrastructure of HLMVEC. Uexposed to air (21% O2) for 2 days or hyperoxia (95% O2) for 8 days. Original magendoplasmic reticulum. For experimental details, see Materials and methods.

of cells exposed to either 21% O2 for 2 days or 95% O2 for8 days. Making comparisons at these respective time pointsallowed for cells in each group to be confluence matched.Compared to control uninfected cells exposed to 21% O2, cellsexposed to hyperoxia showed mitochondrial cristae whichappeared swollen, and the endoplasmic reticulum was dis-tended. By contrast, the myrAkt-transduced cells lackedswollen cristae in mitochondria relative the LacZ-transduced

ntransduced, Ad-LacZ-transduced, and Ad-myrAkt-transduced HLMVEC werenification, ×31,000. Bar at the bottom indicates 500 nm. M, mitochondria; ER,

Fig. 8. Morphometric analyses of mitochondria and endoplasmic reticulum in HLMVEC exposed to hyperoxia. Untransduced, Ad-LacZ-transduced, and Ad-myrAkt-transduced HLMVEC were exposed to air (21% O2) for 2 days or hyperoxia (95% O2) for 8 days. Morphometric analysis of volume density (top) and volume-to-surface area (bottom) was performed and plotted as detailed under Materials and methods. *Statistical difference from the control cells in air.


or nontransduced control cells exposed to hyperoxia. Also theendoplasmic reticulum was better preserved in the myrAkt-transduced cells exposed to hyperoxia. Morphometric analysisof mitochondria showed a decrease in both the volume densityand the volume-to-surface area in the myrAkt-transduced cellsexposed to hyperoxia compared with the two types of controls(Fig. 8). There was a similar decrease in volume-to-surface areaof endoplasmic reticulum in the myrAkt-transduced cellsexposed to hyperoxia compared to the control cells.

Discussion

Exposure to elevated concentrations of inspired oxygen(hyperoxia) can cause activation of signaling pathways. In thepresent study we have established a protective role for Aktactivation during hyperoxic exposure. Increased Akt phosphor-ylation was apparent at 1 and 24 h but declined by 48 h ofhyperoxic exposure (Fig. 1). Phosphorylation of Akt could bedue to direct activation of PI3-kinase by ROS produced as aresult of hyperoxia or due to reversible inactivation of PTEN[38]. Activation of Akt mediated through ROS also occurs inother systems, including the circulatory system [39].

In an effort to understand the significance of Akt activation,we determined the effects of transducing cells with active Aktbefore hyperoxic stress. Overexpression of constitutively activeAkt decreased cell death relative to controls during hyperoxia(Figs. 2 and 6). This finding supports a protective role for Akt in

hyperoxic injury of the endothelium. In addition, it suggests thatAkt can protect against necrotic cell death. Nuclear morphologyis one indicator of the type of cell death process. Nuclearswelling is a characteristic of necrotic cell death [31]. Whilepreserving cell integrity, Akt overexpression also preservednuclear morphology.

An early increase in Akt phosphorylation in response tooxidative stress that occurs during hyperoxia could signaladaptive pathways to limit lung injury. We have shownpreviously that brief hyperoxic exposures cause extracellularATP release [32]. In addition, extracellular ATP can increasePI3-kinase and p70 S6-kinase activities [32,40]. Early eventssuch as ATP release and elevation of glucose uptake in responseto hyperoxia may facilitate survival. Similarly, endothelial Aktactivation by fluid shear stress has been reported to suppressapoptosis [41]. Other cell stressors, including hypoxia [20] ortreatment with N-methyl-D-aspartate, hydrogen peroxide, oretoposide [42], also can activate Akt. The activation of Akt bysuch a broad range of oxidants and potential toxins indicatesthat it may function as a redox sensor, setting in motiondownstream protective pathways.

The prolonged increase in Akt activity during the initial 24h of hyperoxic exposure (Figs. 1B and 1C) could signaldownstream survival pathways to limit injury. However, at 48h of hyperoxic exposure, both Akt phosphorylation and totalAkt levels decrease, indicating a shift away from the prosurvivalpathway. These findings in HLMVEC contrast those previously


reported in the human lung adenocarcinoma cell line A549. InA549 cells, Akt phosphorylation does not remain elevated at 24h of hyperoxic exposure [43]. This is perhaps understandablebecause, unlike the primary HLMVE cells used in this study,A549 cells have a high constitutive level of PI3-kinase activityand active Akt, a characteristic of many cancer cells.

A prolonged increase in Akt phosphorylation also occursduring hypoxic exposure for 24 h (Fig. 1B). Prior exposure tohypoxia for 24 h is protective against a subsequent hyperoxicinsult in several cell types [21]. In addition, pretreatment ofeither A549 cells or HLMVEC with PI3-kinase inhibitorsduring hypoxia, when PI3-kinase activity normally is increased,enhances hyperoxic cell death [21]. These observations lead usto believe that Akt also could mediate hypoxic preconditioning.

Lu et al. [24] demonstrated that targeted delivery of myrAktto lung epithelium diminished acute lung injury and death dueto hyperoxia, but the mechanisms responsible for suchprotection remained incompletely understood. In a relatedstudy, targeted delivery of keratinocyte growth factor to micelung epithelium protected the epithelium, but not the endothe-lium, against hyperoxic death through an Akt-dependentpathway [44]. Importantly, protection of the epithelium didnot increase the survival of mice exposed to hyperoxia,underscoring the importance of endothelium in hyperoxicinjury and survival [44]. Previously, that the microvascularendothelium is a principal early target of hyperoxic injury hasbeen shown in rodents [4,45] and primates [26,27]. The presentstudy defines several events downstream of Akt activation inthese critical susceptible cells.

Hyperoxia causes inactivation of tricarboxylic acid cycleenzymes, especially aconitase [6]. The reduced activity of theTCA cycle enzymes and associated generalized decline inmitochondrial respiration necessitate greater cellular depen-dence upon glycolysis for energy homeostasis. Glucoseconsumption by cells exposed to hyperoxia gradually increasesduring the initial exposure period [7], concomitant with thedecline in aconitase and respiration [6]. Because Akt canupregulate glucose utilization, it was reasonable to speculatethat cells transduced with myrAkt would have altered glucosemetabolism. With transient Akt activation after hyperoxicexposure, one might expect glucose uptake to increase.However, we did not find an increase in 2-deoxyglucose uptakeafter 60 min of hyperoxic exposure in any cell. Enhancement inthe uptake of 2-deoxyglucose occurs only after more prolongedexposure to hyperoxia [32], suggesting that the gene activationand/or protein expression responsible require N1 h [46].Nonetheless, there was a significant increase in basal glucoseuptake of cells transduced with myrAkt relative to controls. It isimportant to emphasize that after 1 h of exposure to hyperoxia,myrAkt-transduced cells already had an elevated Akt expres-sion mediated by transduction with the Ad vector. Thus,pathways responsible for increased glucose utilization alreadywere “primed” or activated by Akt before exposure of the cellsto hyperoxia.

As part of a normal adaptation, exposure to hyperoxia leadsto gradually increased glucose consumption during the first 48h [7]. Clearly myrAkt expression led to an additional increase in

glucose consumption in HLMVEC during exposure tohyperoxia. Akt-mediated increased glucose uptake and utiliza-tion in cells could occur through its downstream targets mTOR(mammalian target of rapamycin) and p70 S6-kinase.

The role of mTOR in cellular energy homeostasis isbeginning to be appreciated [47]. Our studies showed thatrapamycin blocked Akt-mediated elevation of glucose con-sumption by cells, under conditions of both air and hyperoxia(Fig. 4), suggesting an mTOR/p70S6K-dependent effect.Additional evidence for an mTOR/p70S6K-dependent pathwayis supported by increased activity of mTOR and p70 S6-kinaseafter Akt transduction as assessed by Western blot usingantibodies against their respective phosphorylated forms (Fig.5). Other than its principal role in regulating translation, p70 S6-kinase also can regulate glucose metabolism as demonstrated bythe glucose intolerance of an S6K−/− mouse [48]. The hyperoxicincrease in glucose consumption of HLMVEC was notcompletely inhibited by treatment with rapamycin (Fig. 4),raising the possibility that additional pathways regulate glucoseconsumption in HLMVEC exposed to hyperoxia.

Cell death often is accompanied by loss of mitochondrialintegrity and function [49]. Lungs exposed to hyperoxia cangenerate ROS including O2

U− and H2O2, and mitochondria arean important source of these ROS [5]. Mitochondria frompulmonary microvascular endothelial cells are both generatorsand targets of these ROS [50]. Our ultrastructural findingsdemonstrated relative preservation of mitochondrial morphol-ogy in cells transduced with Ad-myrAkt relative to control cellsin hyperoxia. Interestingly, the mitochondria from A549 cells[10], lung-derived, epithelial-like adenocarcinoma cells, ex-posed to hyperoxia appeared much larger than the mitochondriafrom the normoxic controls. However, in HLMVEC there wasinstead an increase in volume of the mitochondrial intracristaespace and not the overall mitochondrial size. Interestingly,morphometric analysis showed an increase in both volumedensity and volume-to-surface area of mitochondria in cellsexposed to hyperoxia, and Akt reversed both these effects.

In endothelial cells, the endoplasmic reticulum, in addition tofunctioning in the synthesis and modification of proteins, acts asa repository for about 75% of the total intracellular calciumreserve [51]. The distended endoplasmic reticulum may suggestaltered intracellular calcium or dysfunctional protein synthesisand/or assembly. This was in contrast to earlier findings inwhich there was an increased number of rough endoplasmicreticulum profiles in granular pneumocytes (type II epithelialcells) of rat lung after 96 h of sublethal hyperoxic exposure [52].Structural differences in the endoplasmic reticulum likely aredue to cell-type differences in susceptibility to oxidative stress.A greater than 50% reduction in microvascular endothelial celland capillary surface area occurs even with sublethal hyperoxicexposure in vivo [25]. By contrast, granular pneumocytesremain viable and even proliferate under these conditions [53].Akt-dependent increased structural integrity of mitochondriaand increased glucose metabolism of cells exposed to hyperoxiasuggest that improved function of energy-producing systemsmay have contributed to the protection observed in these cells.This is supported by findings of other investigators who have


shown that glucose availability coupled to its metabolism isimportant in Akt-mediated survival and preservation ofmitochondrial integrity [18].

Although themselves of interest within the pulmonarymicrocirculation, the findings reported here may have perti-nence and parallels in the coronary circulation. Oxidative stressis a unifying theme in the pathogenesis of cardiovasculardisease [54]. As in the lung, comparative studies indicate thatthe coronary endothelial cell is even more sensitive to oxidativemitochondrial damage than cardiomyocytes [55,56]. In addi-tion, excessive production of reactive oxygen and nitrogenspecies is an essential feature in the pathogenesis of protectionby cardiac preconditioning against subsequent ischemia–reperfusion injury [22,57]. Several recent studies indicate thatthe PI3k/Akt pathway also can be involved in this process.Thus, these findings are potentially also relevant to the cardiaccirculation.

To our knowledge this is the first report demonstrating Aktactivation due to hyperoxia. This study suggests that briefoxygen therapy could trigger beneficial responses to promotecell survival and diminish toxicity. Further, this study showsthat Akt is an important signal mediating mTOR-dependentupregulation of glucose consumption and associated cellsurvival in hyperoxia. In addition to its role in protectingagainst apoptotic cell death shown in other studies, this studysuggests that Akt also protects against necrotic cell death inoxidative injury and preserves mitochondrial structure. Resultsfrom the current investigation along with our previous studiesfurther support an important protective role of the PI3k/Akt/mTOR/p70S6K pathway in hyperoxia.

Acknowledgments

This work was supported by National Institutes of HealthGrant HL56263 (C.W.W.), Colorado Tobacco Research Pro-gram Grant 3R-020 (C.W.W.), U.S. Environmental ProtectionAgency Grant R82570201 (C.W.W.) and grant numberES014448-01 from the National Institute of EnvironmentalHealth Sciences (NIEHS) (C.W.W.). A. Ahmad was a recipientof the Florence Myers Goldhamer Fellowship in PediatricsAllergy and Immunology funded by the National JewishMedical & Research Center (Denver, CO). We thank GretchenHugen for preparation of the manuscript.

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endothelial akt activation by hyperoxia: role in cell survival

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