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Published ahead of print on July 13, 2007, doi:10.1165/rcmb.2006-0429OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 37, pp. 699-705, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2006-0429OC

Stretch-Induced Alveolar Type II Cell Apoptosis

Role of Endogenous Bradykinin and PI3K-Akt Signaling

Stefan Hammerschmidt1, Hartmut Kuhn1, Christian Gessner1, Hans-Jurgen Seyfarth1 and Hubert Wirtz1

1 Department of Respiratory Medicine, University of Leipzig, Leipzig, Germany

Correspondence and requests for reprints should be addressed to PD Dr. Stefan Hammerschmidt, Medizinische Universitätsklinik I, Pneumologie, Universität Leipzig, Johannisallee 32, 04103 Leipzig, Germany. E-mail: stefan.hammerschmidt{at}t-online.de


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Apoptosis of alveolar type II (ATII) cells in response to high-amplitude mechanical stretch represents an important mechanism of ventilation-induced lung injury. Previously, it was demonstrated in an in vitro model that stretch-induced ATII cell apoptosis was prevented by angiotensin-converting enzyme (ACE) inhibitors. This study investigates the mechanism by which ACE inhibitors prevent stretch-induced apoptosis and elucidates the role of bradykinin as an endogenous anti-apoptotic factor. Rat ATII cells cultured on flexible membranes were subjected to cyclic stretch (40 cycles/min; 30% increase in surface area) and compared with static controls. Angiotensinogen, the bradykinin precursor T-kininogen, and bradykinin receptor expression were measured by RT-PCR; Angiotensin II and phosphoinositol 3 OH-kinase (PI3K) activity (as phospho-Akt) were measured by enzyme-linked immunosorbent assay; and Bcl-2 and Bcl-XL were measured by Western blot. Stretch did not influence angiotensinogen expression or induce angiotensin II generation. The angiotensin II receptor antagonist saralasin did not prevent stretch-induced apoptosis, whereas ACE inhibitors did. Stretch reduced ATII cell bradykinin release (T-kininogen expression and bradykinin supernatant concentration), and subsequently led to reduced PI3K activity and decreased concentrations of the anti-apoptotic proteins Bcl-2/Bcl-XL. Bradykinin substitution or addition of keratinocyte or hepatocyte growth factor prevented stretch-induced decrease in PI3K activity and Bcl-2/Bcl-XL and reduced stretch-induced apoptosis. Mechanical stretch impairs a constitutively expressed, autocrine anti-apoptotic ATII cell survival signal involving bradykinin-mediated stimulation of the PI3K–Akt–Bcl-2/Bcl-XL pathway. Restoration of this pathway prevents stretch-induced apoptosis. This may be beneficial when mechanical ventilation cannot completely avoid alveolar overdistension to maintain oxygenation.

Key Words: mechanical stretch • apoptosis • PI3K • bradykinin



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Mechanical stretch impairs alveolar epithelial cell anti-apoptotic survival signaling via PI3K–Akt–BAD–Bcl-2/Bcl-XL. Restoration of PI3K activity prevents stretch-induced apoptosis and increases the tolerance of the cells to mechanical stretch.

 
The use of low tidal volumes has improved outcome in acute respiratory distress syndrome (1). This strategy recognizes that high tidal volume ventilation aggravates or causes acute lung injury by overdistension-induced damage inflicted on pulmonary cells. Distension of a presumed physiologic extent in alveolar cells has been associated with calcium signaling and phospholipid secretion (2) and with surfactant protein expression (3). However, high-amplitude mechanical stretch induces cell membrane stress failure (4), resulting in cell death (5) and apoptosis (5–8). Due to the inhomogeneous nature of most lung injuries, ventilation with a low tidal volume in injured lungs may still result in alveolar overdistension because of derecruitment of alveolar units and an increased risk of overdistending the remaining open lung units. Even moderate mechanical stretch leading to an increase in cell surface area of 30% and a stretching frequency of 40 cycles per minute, simulating lung inflation to just about 100% total lung capacity were sufficient to induce alveolar type II (ATII) cell apoptosis and necrosis in a cell culture model (8). This moderate level of alveolar cell overdistension cannot be entirely avoided. Therefore it appears important to investigate mechanisms of protection against distension-induced cell damage.

The angiotensin-converting enzyme (ACE) inhibitor captopril has been shown to prevent mechanical stretch–induced apoptosis of ATII cells in our model (8). ACE inhibition is widely used for congestive heart failure and arterial hypertension and in this context has also been recognized as an anti-apoptotic intervention (9).

ACE inhibitors and angiotensin II receptor antagonists exert anti-apoptotic effects on pulmonary epithelial cell apoptosis (10–13). These effects have been interpreted as evidence for a local renin-angiotensin system (14). In our model we could not detect angiotensin II generation in cyclically stretched cells as an indicator of the action of a local renin-angiotensin system. Therefore we looked for alternative mechanisms by which ACE inhibition may prevent mechanical stretch–induced apoptosis.

ACE inhibitors are also known to prevent the degradation of bradykinin (15), a peptide that is produced by kallikrein induced proteolytic cleavage of kininogens. ACE inhibition may therefore lead to an increase in local bradykinin concentrations. Bradykinin has been shown to have anti-apoptotic and protective effects mediated by the bradykinin subtype 2 receptor (16, 17). It has been suggested that bradykinin subtype 2 receptor signaling may involve phosphoinositol 3 OH-kinase (PI3K) activation and subsequent phosphorylation of Akt kinase (16, 18, 19). Activated Akt (pAkt) in turn has been shown to reduce apoptosis in a human pulmonary endothelial cell line due to the inactivation of pro-apoptotic Bad by phosphorylation at Ser-136 (20). Phosphorylation of Bad inactivates its proapoptotic function by dissociating Bad from the anti-apoptotic proteins Bcl-2 and Bcl-XL (21, 22).

This study was performed to investigate the mechanism by which ACE inhibition prevents overdistension-induced ATII cell apoptosis. We have focused the study on two potential actions of ACE inhibitors. (1) Overdistension-induced apoptosis of ATII cells involves the activation of a local renin-angiotensin system. ACE inhibition prevents overdistension-induced ATII cell apoptosis by inhibition of the conversion of angiotensin I into angiotensin II. (2) ACE-inhibition prevents the proteolysis of bradykinin and increases bradykinin concentration. Bradykinin exerts anti-apoptotic effects via PI3K and Akt kinase signaling.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
ATII Cell Preparation
ATII cells were prepared according to the method developed by Dobbs and coworkers (23) as described previously (2). In brief: ATII cells were isolated from male Sprague-Dawley rats (150–200 g) by elastase digestion and differential adherence on IgG-coated dishes. ATII cells were 90 ± 4.7% pure at the time of plating, as proven by modified Papanicolaou staining.

ATII cells were placed on the central area (~ 1.5 cm diameter) of fibronectin-coated silicon membranes (Bioflex, coated additionally with 150 µM bovine fibronectin for at least 3 h at 4°C; Flexcell International, Hillsborough, NC) of six-well plates (106 per well with Dulbecco's modified Eagle's medium [DMEM] with 10% fetal calf serum [FCS], 1%wt/vol gentamicin, and 1% glutamine). After 22 hours of adherence, medium was replaced by fresh medium containing 2% FCS. The medium used in the experiments contained all compounds added for specific experiments (captopril [100 ng/ml], bradykinin [20–2,000 nM], keratinocyte growth factor [KGF] [10 ng/ml] and hepatocyte growth factor [HGF] [50 ng/ml], and saralasin [0.1–10 µg/ml]).

These plates were used for the experiments.

Experimental Protocol
ATII cells plated on Bioflex plates (106 each) were cyclically stretched using the FX 4000T Flexercell Tension Plus system (Flexcell International). The stretching pattern (Stretch-group) was characterized by a frequency of 40 cycles/minute and an increase of 30% in surface area of the silicon membranes as described previously (8). This stretching pattern simulates the change in alveolar basement membrane area during lung inflation to roughly 100% of total lung capacity (24) and has been demonstrated to induce considerable ATII cell apoptosis (8). Membrane distension was calibrated with negative pressure for each instrument and type of membrane and monitored during the experiment. ATII cells, on identical fibronectin-coated silicon membranes not subjected to cyclic distension, served as static controls (Static group). The stretching pattern was applied over the entire experimental time. Cells and supernatants were collected at 24 hours unless stated otherwise.

LDH Release
Supernatants of stretched cells and controls were analyzed for lactic acid dehydrogenase (LDH) activity. LDH activity was measured by use of a Cytotoxicity Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany). The kit was calibrated using LDH standards (Sigma-Aldrich GmbH, Deisenhofen, Germany) between 2 and 0.016 µg/ml. Each value represents seven cell isolations with measurements in triplicate.

Apoptosis, Necrosis
Analysis of cell pellets for apoptosis and necrosis has previously been described (8). Trypsin-EDTA (Invitrogen, Paisley, Scotland) was used to harvest the cells for analysis. Cells were incubated with trypsin (0.05%) EDTA (0.2 g/L) for 3 to 4 minutes. We have found that ATII cell viability was not affected by incubation with trypsin EDTA for 2 to 10 minutes.

The cells were harvested, stained with FITC-conjugated Annexin V and propidium iodide (PI) using the Apoptosis Detection Kit (R&D Systems, Minneapolis, MN), and analyzed by flow cytometry (Epics XL; Beckman-Coulter, Krefeld, Germany). Both adherent and nonadherent cells were included in the analysis.

Each value represents seven cell isolations with measurements in triplicate. In the dose–response relations, each data point represents measurements from n = 5 ATII cell isolations.

Enzyme-Linked Immunoassays
We tried to detect angiotensin II by two specific immunoassays (Angiotensin II Detektion Kit; JBL, Hamburg, Germany; and Angiotensin II EIA Kit; Cayman Chemicals, Ann Arbor, MI). Bradykinin was measured using the Bradykinin EIA (Peninsula Laboratories, San Carlos, CA).

Phospho-Akt was measured with a specific enzyme-linked immunosorbent assay (ELISA) (Phospho-Akt [Ser473] Pathscan Sandwich ELISA Kit; Cell Signaling Technology, Danvers, MA) in cell lysates. Phospho Akt concentrations were analyzed in samples of equivalent protein concentrations. Each value represents seven cell isolations with measurements in duplicate.

Microarray Analysis
Microarray analysis was performed at a certified facility of the University of Leipzig according to the Affymetrix GeneChip protocol (Affymetrix, Santa Clara, CA). The microarray analysis was performed twice for each condition (static cells and stretched cells). One assay contained RNA of three separate experiments. Total RNA was isolated from harvested cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Ten micrograms of total RNA were used to prepare double-stranded cDNA (Superscript II; Life Technologies, Gaithersburg, MD) primed with oligo-dT containing a T7 RNA polymerase promoter site (Genset SA, Paris, France). cDNA was purified by phenol-chloroform extraction before in vitro transcription using the ENZO BioArray RNA transcript labeling kit (Affymetrix) to synthesize biotin-labeled cRNA. After transcription, unincorporated nucleotides were removed using the RNeasy kit (Qiagen). The cRNA was fragmented and hybridized to Affymetrix GeneChip U95Av2 at 45°C for 16 hours. Washing, staining, and scanning of the probe array was performed according to the manufacturer's instructions. Raw data were scaled to a target intensity of 150 using Microarray-Suite Software 5.0 (Affymetrix).

The differences of gene expression between static and stretched cells were calculated using the batch-analysis function (MAS 5.0). Data of the static cells served as a baseline. Data labeled as present were selected, and filtered with Excel software (Microsoft, Redmond, WA) (signal log ratio [>= 0.5/<= –0.5]; change-P value [< 0.005/> 0.995]).

Real-Time RT-PCR Analysis of Angiotensinogen, T-Kininogen, Bradykinin Receptor 1 and 2 Subtype
RNA was isolated from harvested cells using the RNeasy Mini Kit (Qiagen). 1µg RNA was digested with DNase (GIBCO/Invitrogen, Karlsruhe, Germany) at room temperature for 15 minutes. Digested RNA was reverse transcribed at 42°C for 30 minutes (Reverse Transcription System; Promega GmbH, Mannheim, Germany).

Primers, annealing temperatures, and the size of the PCR product of the genes of interest and a GAPDH control are shown in supplementary Table 1. PCR was performed in a final volume of 20 µl, with 1 µl cDNA, 10 µl iQ SYBR Green Supermix (Bio-Rad, Hercules, CA), and 9 µl H2O. PCR was performed with an initial denaturation at 94°C for 3 minutes, cycling times of 30 seconds denaturing at 94°C, 30 seconds annealing, and 30 seconds extension at 72°C and final extension at 72°C for 7 minutes. PCR-Products were detected during PCR with the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad). Real-time PCR data were automatically calculated with the data analysis module. The results were analyzed according to the {Delta}{Delta}Ct method (25). Real-time PCR was performed in triplicate with cDNA from four cell isolations.


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  		TABLE 1. PRIMER, ANNEALING TEMPERATURE, AND PCR PRODUCTS (BASE PAIRS) OF THE GENES OF INTEREST AND OF GAPDH AS INTERNAL STANDARD

 
Western Blot Analysis of Bcl-2 and Bcl-XL
Cell extracts were lysed on ice (50 mM HEPES pH 7.5, 200 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 0.1% Tween-20, 10 mM beta-glycerophosphate, 1 mM DTT, 0.1 mM PMSF, 5 µg/ml leupeptin). Protein concentrations were determined by DC Protein Assay (Bio-Rad). For Western blotting, 10 µg protein per lane were resolved by SDS-PAGE and transferred onto nitrocellulose membranes (Hybond-N; Amersham/GE Healthcare, Buckinghamshire, UK) by standard procedures. Primary monoclonal antibodies against Bcl-XL (Clone 44) and Bcl-2 (Clone 7) were purchased from BD Biosciences/PharMingen (Rockville, MD) and the antibody against GAPDH (6C5) from HyTest Ltd. (Turku, Finland). The HRP labeled secondary antibody was purchased from Sigma. ECL (Amersham/GE Healthcare) was used for detection.

Kallikrein Enzyme Activity
Supernatants were incubated with substrate (Pro-Phe-Arg-methylcoumarylamide) in 0.1 M Tris-HCl buffer containing 0.15 M NaCl, at pH 8.0. Hydrolysis of the substrate was recorded for the first 5-minute period. Amino-methylcoumarin is generated by Pro-Phe-Arg-methylcoumarylamide hydrolysis. Amino-methylcoumarin was detected by fluorescence at (excitation 370 nm/emission 460 nm). One unit of kallikrein activity releases 1 nM amino-methylcoumarin in 5 minutes. Each data point represents five cell isolations with measurements in triplicate.

Statistics
Data are mean ± SD. Experimental groups were compared using ANOVA and post hoc Bonferroni analysis or by the Student's t test (SPSS 11.0; SPSS Inc., Chicago, IL).


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
ACE Inhibition, Angiotensin II Generation, Angiotensin II Receptor Antagonist
In the first set of experiments, we investigated whether cyclic mechanical stretch of ATII cells results in angiotensin II generation. We examined ATII cell gene expression of angiotensinogen, the precursor of angiotensin II, as well as angiotensin II concentrations in supernatants of ATII cell cultures after 24 hours of cyclic stretch. Mechanical stretch did not alter angiotensinogen gene expression compared with static controls (Table 2). Angiotensin II the final product of the proteolytic cleavage of angiotensinogen by renin and ACE was not detectable in the supernatants of static and stretched ATII cells, despite the use of different angiotensin II ELISA test kits (lower limit 1.5 pg/ml).


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  		TABLE 2. GENE EXPRESSION

 
In a second set of experiments, the nonselective angiotensin II receptor antagonist saralasin was then used to investigate the possibility that angiotensin II, in concentrations below the detection limit of the test kits used, would still be involved in stretch-induced ATII cell apoptosis (Figure 1). The effect of saralasin (1 µg/ml) on overdistension-induced apoptosis was compared with the effect of the ACE inhibitor captopril (10 ng/ml) that has been reported previously. In addition, a structurally dissimilar ACE inhibitory compound (Sigma A-0773; 10 ng/ml) was used in this set of experiments to rule out any nonspecific effects of captopril. captopril and the peptide ACE inhibitor, but not the angiotensin II receptor antagonist, prevented mechanical stretch–induced apoptosis. In an additional set of experiments we tested three saralasin concentrations (0.1–10 µg/ml), but also did not find a significant influence on mechanical stretch–induced apoptosis. We therefore concluded that angiotensin II did not appear to be involved in stretch-induced apoptosis in this system, even if it was indeed present in our system in very low concentrations.


Figure 1
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  		Figure 1. Stretch-induced damage of alveolar type II (ATII) cells—influence of angiotensin-converting enzyme (ACE) inhibition and saralasin. Rat ATII cells on flexible membranes were subjected to cyclic stretch (Stretch: frequency 40 cycles/min, 30% increase in cell surface area) or kept under static conditions (Static) for 24 hours. Captopril (10 ng/ml; Cap), the peptide ACE inhibitor (Sigma A-0773; 10 ng/ml; ACE-I), or the nonspecific angiotensin II receptor antagonist saralasin (1 µg/ml; Sar) were added constituents of the medium used. The supernatant was analyzed for LDH concentration (A), the cells were analyzed for propidium iodide (PI) staining and annexin V binding. Annexin V–negative/PI-negative cells were regarded as viable (B), Annexin V–positive cells as apoptotic (C) and Annexin V–negative/PI-positive cells as necrotic (D) as described previously (8). (E–H) Dose–response relations for saralasin are given. *P < 0.05 versus corresponding static control (ANOVA and post hoc Bonferroni analysis). {dagger}P < 0.05 versus stretch without addition of Cap, ACE-I, or Sar (ANOVA and post hoc Bonferroni analysis).

 
Bradykinin Generation and Bradykinin Effects on Stretch-Induced Apoptosis and PI3K Signaling
ACE inhibitors are also known to prevent the degradation of bradykinin (15), a peptide that is produced by kallikrein-induced proteolytic cleavage of kininogens. Gene array experiments of rat ATII cells subjected to 24 hours of static culture or cyclic stretch revealed that one out of four rat kininogen genes, the rat T-kininogen, was expressed in rat ATII cells. Expression of the rat T-kininogen was next analyzed by quantitative RT-PCR and found to be decreased to 55% in response to 24 hours of mechanical stretch compared with static controls (Table 2). Supernatant kallikrein activities and bradykinin concentrations as well as the percentage of apoptotic cells after 24 hours of mechanical stretch or static culture are demonstrated in Figure 2. Mechanical stretch, when compared with static controls, led to significant increases in kallikrein activity of culture supernatants, regardless of added captopril or bradykinin. Bradykinin concentrations in supernatants of stretched cultures at the same time were significantly reduced by approximately 25% compared with static cultures. Importantly, this stretch-induced decrease in supernatant bradykinin concentration was not observed when captopril was added to the medium. In static controls, however, captopril did not increase supernatant bradykinin concentrations.


Figure 2
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  		Figure 2. Apoptotic cell death, supernatant kallikrein activity, and bradykinin concentrations. Rat ATII cells on flexible membranes were subjected to cyclic stretch (Stretch: frequency 40 cycles/min, 30% increase in cell surface area) or kept under static conditions (Static) for 24 hours. Captopril (10 ng/ml; Cap) or bradykinin (200 nM; Bra) were added constituents of the medium used. The supernatants were analyzed (A) for kallikrein activity and (B) for bradykinin concentration (except when additional bradykinin was added). Cells were analyzed by flow cytometry for Annexin V binding. (C) The percentage of Annexin V–positive cells was noted for the experimental groups. (D) A dose response indicating the percentage of apoptotic cells in response to different bradykinin concentrations is given. *P < 0.05 versus corresponding static control (ANOVA and post hoc Bonferroni analysis). {dagger}P < 0.05 versus stretch without addition of Cap or Bra (ANOVA and post hoc Bonferroni analysis).

 
Finally, we examined the influence of added bradykinin on stretch-induced apoptosis. Bradykinin is rapidly inactivated due to proteolytic degradation in serum (15). Therefore, we tested the stability of bradykinin in our culture medium containing 2% FCS and found measurable bradykinin concentrations at 24 hours with initial bradykinin concentrations of 20 µM or higher. We decided to substitute bradykinin in concentrations below this and above the bradykinin supernatant concentrations measured. The addition of bradykinin prevented stretch-induced ATII cell apoptosis in a dose-dependent manner.

We also investigated gene expression of the bradykinin receptors after 24 hours of cyclic stretch or static culture (Table 2). Possibly in reaction to decreased bradykinin concentrations, the bradykinin subtype 2 receptor was found significantly up-regulated in cyclically stretched cell cultures.

There are reports of anti-apoptotic and protective effects of bradykinin mediated by the bradykinin subtype 2 receptor subtype in other systems (16, 17). These effects involve PI3K activation with subsequent phosphorylation of Akt kinase (protein kinase B) (16, 18, 19), which in turn phosphorylates and inactivates the pro-apoptotic protein Bad at Ser-136 (20), resulting in Bad dissociation from the anti-apoptotic proteins Bcl-2 and Bcl-XL (21, 22).

Figure 3A indicates a continuous decrease in pAkt content in stretched cells compared with static cells during the 24-hour experimental period. This decrease of pAkt was partially prevented by the ACE inhibitor captopril (10 ng/ml), and more so by direct addition of bradykinin (200 nM, Figure 3B). Figure 3C demonstrates decreased Bcl-2 and Bcl-XL content in cells subjected to 24 cyclic stretch compared with static controls. The decrease of these anti-apoptotic proteins was found to be markedly lower in cyclically stretched cells after addition of captopril or bradykinin (Figure 3C). Bradykinin decreases slightly the Bcl-2 expression (but not Bcl-XL expression) in static controls compared with untreated or captopril-treated cells.


Figure 3
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  		Figure 3. Phosphoinositol 3-OH kinase signaling. Rat ATII cells on flexible membranes were subjected to cyclic stretch (Stretch: frequency 40 cycles/min, 30% increase in cell surface area) or kept under static conditions (Static) for 24 hours. (A) Cell pellets were obtained after 10 minutes, 30 minutes, 6 hours, and 24 hours and analyzed for pAkt content. A exhibits the time course of stretched cells (open squares) compared with static controls (solid squares). (B) The panel demonstrates 24-hour values of experimental groups (duplicate measurement of n = 7 ATII cell preparations) without any additional substance and with addition of captopril (10 ng/ml; Cap) or bradykinin (200 nM; Bra). (C) The panel shows representative western blots of Bcl-2, Bcl-XL, and GAPDH. Cells subjected to cyclic stretch (Stretch) are compared with static cultures (Static). Experiments without supplement (–) to the medium are compared with experiments with addition of captopril (Cap) or bradykinin (Bra). *P < 0.05 versus static control; {dagger}P < 0.05 versus corresponding static control and versus Stretch (ANOVA and post hoc Bonferroni analysis).

 
PI3K Activation and Overdistension-Induced Apoptosis
Finally, we tested whether other compounds—like bradykinin—stimulating the PI3K–Akt–Bcl-2/Bcl-XL pathway also prevent stretch-induced apoptosis. Growth factors such as KGF and HGF are known to stimulate PI3K-Akt signaling in ATII cells, and have been reported to inhibit apoptosis and to stimulate cell proliferation and differentiation by stimulating the PI3K–Akt signaling pathway (26, 27) reduce the susceptibility to stretch-induced cell death (28).

In our model, both KGF (10 ng/ml) and HGF (50 ng/ml) led to an increase in cellular pAkt in cyclically stretched cells (Figure 4A) and to reduced stretch-induced apoptosis (Figure 4B) at 24 hours of cyclic stretch.


Figure 4
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  		Figure 4. HGF and KFG—effect on PI3K activity and ATII cell apoptotic cell death. Rat ATII cells on flexible membranes were subjected to cyclic stretch (Stretch: frequency 40 cycles/min, 30% increase in cell surface area) or kept under static conditions (Static) for 24 hours. HGF (50 ng/ml) or KGF (10 ng/ml) was a constituent of the medium used. At 24 hours, cells were analyzed (A) for pAkt content by ELISA and (B) for Annexin V surface expression by flow cytometry. *P < 0.05 versus static control; {dagger}P < 0.05 versus corresponding static control and versus Stretch (ANOVA and post hoc Bonferroni analysis).

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Previously, we reported that cyclic mechanical stretch with a 30% increase in cell surface area causes apoptosis in approximately 30% of the ATII cells subjected to this stretching pattern. Apoptosis has been demonstrated by Annexin V binding, release of oligonucleosomen, and caspase activation (8). A stretching amplitude of 30% increase in surface area corresponds to the extent of deformation of the alveolar basement membrane reached during lung inflation to 100% of total lung capacity. This extent of mechanical stretch therefore does not greatly exceed the physiologic range. It may well occur or even be exceeded in units of injured, inhomogeneous lungs during mechanical ventilation, albeit ventilation is performed according to a "protective" protocol. Overdistension-induced apoptosis cannot be completely avoided when even minimal ventilatory needs have to be fulfilled, and preventing stretch-induced apoptosis by means other than avoiding ventilation appears to be an attractive option to improve outcome in patients under mechanical ventilation due to acute lung injury.

Alveolar epithelial and A549 cell apoptosis in response to several stimuli, including Fas-Ligand or Fas-stimulating antibodies (10, 11), amiodaron, and bleomycin (12, 13) has been prevented by ACE inhibition and by angiotensin II receptor antagonists. In addition, increased gene expression of angiotensinogen and the generation of angiotensin II has been shown to occur in Fas-Ligand induced apoptosis of alveolar epithelial cells (11). Antisense oligonicleotides against angiotensinogen inhibited Fas ligand–induced apoptosis and angiotensin II–induced apoptosis in alveolar epithelial cells (11, 29). This observation suggested the presence of a local renin-angiotensin system that contributes to alveolar epithelial cell apoptosis in response to injurious stimuli (14). Mechanical stretch–induced ATII cell apoptosis in our model is also reduced by the ACE inhibitor captopril (8). We therefore hypothesized that mechanical stretch–induced apoptosis of ATII cells involves the activation of a local renin-angiotensin system, including expression of angiotensinogen and its conversion into angiotensin I and angiotensin II.

Several approaches, however, failed to support this hypothesis. (1) We did not find changes in angiotensinogen expression between stretched and static ATII cells. (2) Although we used two different angiotensin II detection kits, we did not detect angiotensin II in supernatants of stretched and static cells. The detection limits of the assays were considerably lower than the pro-apoptotic concentration of angiotensin II (EC50 between 10 and 50 nM, i.e., between 10.5 and 52.5 pg/ml) reported in the literature (29). (3) The nonselective angiotensin II receptor antagonist saralasin did not cause significant changes in mechanical stretch–induced apoptosis in concentrations from 0.1 to 10 µg/ml with proven anti-apoptotic activity in other models (0.05 to 50 µg/ml (11)). This indicates that angiotensin II concentrations below the detection limit of the ELISA kits were not involved in stretch-induced apoptosis.

We conclude that ACE inhibitors prevented stretch-induced apoptosis by means other than the inhibition of angiotensin I conversion to angiotensin II.

ACE inhibition also inhibits the proteolytic degradation of the peptide bradykinin (15). Bradykinin may evoke protective and anti-apoptotic effects after the activation of PI3K with subsequent phosphorylation and activation of the anti-apoptotic Akt kinase in other models (16–19). Therefore, we investigated bradykinin generation and possible sequels in our model of mechanical stretch–induced ATII cell apoptosis. We observed reduced expression of the bradykinin precursor T-kininogen and subsequently a reduction of supernatant bradykinin concentrations in response to mechanical stretch although the activity of the bradykinin-generating enzyme kallikrein was found to be increased in response to mechanical stretch. Our interpretation therefore involves a constitutive generation of T-kininogen and a release of bradykinin from ATII cells that is impaired by cyclic high-amplitude mechanical stretch. Inhibition of bradykinin decay by captopril in turn restores bradykinin concentrations in the supernatant of ATII cells subjected to cyclic stretch to a level comparable with that in static cell cultures. In addition, added bradykinin prevented cyclic mechanical stretch–induced apoptosis in ATII cells. This points out the significance of the fall in ATII cell supernatant bradykinin concentration induced by mechanical stretch.

Bradykinin is a short-lived peptide with a half-life time of some seconds in serum. This short half time is predominately due to proteolytic decay by serum proteases (15). Although the culture medium contained only 2% FCS, a considerable bradykinin breakdown has been demonstrated. Therefore the supernatant bradykinin concentrations are influenced by de novo synthesis and by breakdown. Because bradykinin breakdown is predominantly due to proteolytic enzymes in the culture medium, the differences may be due to differences in bradykinin synthesis and release. Bradykinin breakdown during the experimental time may explain why the differences among the supernatant bradykinin concentrations are rather moderate compared with differences in pAkt. Our observation that captopril does not increase the concentration of bradykinin in the static cells relative to untreated cells cannot be explain by the experiments of this study. A negative feedback that controls bradykinin production under static conditions may represent an explanation.

During acute lung injury the breakdown of the alveolar–capillary barrier facilitates the leak of serum proteins into the alveolar space. Serum proteases may accelerate the breakdown of local alveolar bradykinin and thereby increase alveolar epithelial cell apoptosis and aggravate acute lung injury.

Inhibition of the ACE protease activity represents not the only explanation of increased bradykinin effects in response to ACE inhibitors (15). Due to the short half-life time, bradykinin is thought to be produced and to exert local effects at tissue sites. The various components required for synthesis of bradykinin as well as its receptors are expected to be present in close proximity. Experiments with cells transfected with ACE and the bradykinin subtype 2 receptor indeed demonstrated close proximity of these proteins, permitting ACE–bradykinin subtype 2 receptor crosstalk. In these experiments ACE inhibitors increased the number of cell surface receptors and inhibited receptor desensitization and internalization (30, 31). These mechanisms may also contribute to the ACE inhibitor effect observed in our model.

The up-regulation of the bradykinin subtype 2 receptor in ATII cells subjected to mechanical stretch may underline the relevance of the observed decrease of the bradykinin supernatant concentration. Bradykinin subtype 2 receptor is up-regulated not only in response to mechanical stretch but also after inflammatory stimuli, such as IFN-{gamma} and TNF-{alpha} in A549 cells (32). These data indicate a crucial role of bradykinin and bradykinin signaling via bradykinin subtype 2 receptor after inflammatory or injurious stimuli. Indeed, bradykinin has been shown to have protective effects in ischemic hearts (33). The well-described cardioprotective effects of bradykinin are mediated by activation of PI3K and subsequent stimulation of Akt kinase. Activated Akt kinase (pAkt) has been shown to reduce apoptosis in a human pulmonary endothelial cell line due to the inactivation of pro-apoptotic Bad by phosphorylation at Ser-136 (20). Phosphorylation of Bad inactivates its proapoptotic function by dissociating Bad from the anti-apoptotic proteins Bcl-2 and Bcl-XL (21, 22). Decreased bradykinin concentrations with subsequently reduced PI3K–Akt kinase signaling will then result in enhanced apoptosis in stretched cells.

These considerations led us to hypothesize that mechanical stretch may result in decreased cellular PI3K–Akt kinase signaling subsequent to decreased bradykinin concentration and that bradykinin may prevent stretch-induced apoptosis by restoring the PI3K–Akt–Bad pathway. Our data demonstrate that mechanical stretch successively reduced PI3K activity to roughly 10 percent of the static control group level and subsequently reduced the cellular content of the anti-apoptotic Bcl-2 and Bcl-XL. Mechanical stretch–induced effects were partially prevented by addition of bradykinin or captopril. These data provide further evidence that reduced bradykinin generation and, in turn, reduced bradykinin receptor–mediated PI3K–Akt–Bcl-2/Bcl-XL signaling in response to cyclic distension are connected to increased apoptosis. Bradykinin substitution both stimulates the PI3K–Akt–Bad pathway and reduces distension-induced apoptosis. Our finding that bradykinin slightly decreases Blc-2 but Bcl-XL expression in static controls cannot be explained by our data. Due to the close proximity of ACE and bradykinin subtype 2 receptor, captopril binding to ACE may modify bradykinin subtype 2 receptor signaling (15) and induce a response different from that of only bradykinin substitution.

Mechanical stress impairs the PI3K–Akt–Bad pathway and reduces the cellular content of Bcl-2 and Bcl-XL, two anti-apoptotic proteins that stabilize mitochondria, thereby preventing apoptosis. This may suggest that mechanical stretch–induced apoptosis is predominantly mediated via the mitochondrial pathway. Mitochondria are connected to the cytoskeleton and therefore mechanical stretch may by transmitted to mitochondria and change their permeability. These considerations are supported by data that correlate effects of mechanical stretch to oxidant release from mitochondria (34).

Whereas mechanical stretch–induced apoptosis seems to be mediated via the mitochondrial pathway, Fas ligand–induced apoptosis is executed by the death receptor pathway (35). Our observation that a local renin-angiotensin system might not be involved in our model of mechanical stretch-induced apoptosis does not, therefore, contradict the data discussed above reporting the activation of a local renin-angiotensin system in Fas ligand–induced apoptosis.

The growth factors KGF and HGF are known to stimulate PI3K-Akt signaling in ATII cells, and have been reported to inhibit apoptosis and to stimulate cell proliferation and differentiation by stimulating the PI3K–Akt signaling pathway (26, 27). KGF also reduced ATII cell susceptibility to mechanical stretch (28). In our model of mechanical stretch–induced apoptosis, these growth factors both partially restored PI3K activity and also reduced the susceptibility to mechanical stretch–induced apoptosis. These activators of the PI3K-Akt-signaling pathway therefore confirmed the role of this signaling pathway in cyclic stretch–induced ATII cell apoptosis. Apparently, this pathway is constitutively active in our model of ATII cells in primary culture. Cyclic mechanical stretch leads to increased apoptosis due to decreased stimulation of PI3K–Akt signaling. Our experiments demonstrate that bradykinin, a product of kallikrein enzymatic activity, is reduced in distended cell cultures. Because kallikrein activity is not reduced, the most likely explanation is the reduced expression of T-kininogen, the precursor of bradykinin in stretched cells. To our knowledge this dependency of cellular survival on T-kininogen expression, bradykinin concentration, and bradykinin stimulation of the PI3K–Akt–Bad signaling pathway through bradykinin subtype 2 receptor has not been described previously. It was discovered because the presumed mechanism of ACE inhibitor–mediated inhibition of apoptosis in our model did not hold true. ACE inhibitors as well as other pharmacologic agents influencing the pathway outlined in this set of experiments might be of use in reducing the damage inflicted upon lung cells in mechanical ventilation.


   Footnotes
 
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG).

Originally Published in Press as DOI: 10.1165/rcmb.2006-0429OC on July 13, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form November 17, 2006

Accepted in final form May 31, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

  1. The Acute Respiratory Distress Syndrome Network. Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. N Engl J Med 2000;342:1301–1308.[Abstract/Free Full Text]
  2. Wirtz HR, Dobbs LG. Calcium mobilization and exocytosis after one mechanical stretch of lung epithelial cells. Science 1990;250:1266–1269.[Abstract/Free Full Text]
  3. Sanchez-Esteban J, Cicchiello LA, Wang Y, Tsai SW, Williams LK, Torday JS, Rubin LP. Mechanical stretch promotes alveolar epithelial type II cell differentiation. J Appl Physiol 2001;91:589–595.[Abstract/Free Full Text]
  4. Vlahakis NE, Hubmayr RD. Invited review: plasma membrane stress failure in alveolar epithelial cells. J Appl Physiol 2000;89:2490–2496. (discussion 2497).[Abstract/Free Full Text]
  5. Tschumperlin DJ, Oswari J, Margulies AS. Deformation-induced injury of alveolar epithelial cells. Effect of frequency, duration, and amplitude. Am J Respir Crit Care Med 2000;162:357–362.[Abstract/Free Full Text]
  6. Tschumperlin DJ, Margulies SS. Equibiaxial deformation-induced injury of alveolar epithelial cells in vitro. Am J Physiol 1998;275:L1173–L1183.[Medline]
  7. Edwards YS, Sutherland LM, Power JH, Nicholas TE, Murray AW. Cyclic stretch induces both apoptosis and secretion in rat alveolar type II cells. FEBS Lett 1999;448:127–130.[CrossRef][Medline]
  8. Hammerschmidt S, Kuhn H, Grasenack T, Gessner C, Wirtz H. Apoptosis and necrosis induced by cyclic mechanical stretching in alveolar type II cells. Am J Respir Cell Mol Biol 2004;30:396–402.[Abstract/Free Full Text]
  9. Garg S, Hofstra L, Reutelingsperger C, Narula J. Apoptosis as a therapeutic target in acutely ischemic myocardium. Curr Opin Cardiol 2003;18:372–377.[CrossRef][Medline]
  10. Uhal BD, Gidea C, Bargout R, Bifero A, Ibarra-Sunga O, Papp M, Flynn K, Filippatos G. Captopril inhibits apoptosis in human lung epithelial cells: a potential antifibrotic mechanism. Am J Physiol 1998;275:L1013–L1017.[Medline]
  11. Wang R, Zagariya A, Ang E, Ibarra-Sunga O, Uhal BD. Fas-induced apoptosis of alveolar epithelial cells requires ANG II generation and receptor interaction. Am J Physiol 1999;277:L1245–L1250.[Medline]
  12. Wang R, Ibarra-Sunga O, Verlinski L, Pick R, Uhal BD. Abrogation of bleomycin-induced epithelial apoptosis and lung fibrosis by captopril or by a caspase inhibitor. Am J Physiol Lung Cell Mol Physiol 2000;279:L143–L151.[Abstract/Free Full Text]
  13. Bargout R, Jankov A, Dincer E, Wang R, Komodromos T, Ibarra-Sunga O, Filippatos G, Uhal BD. Amiodarone induces apoptosis of human and rat alveolar epithelial cells in vitro. Am J Physiol Lung Cell Mol Physiol 2000;278:L1039–L1044.[Abstract/Free Full Text]
  14. Fine A, Janssen-Heininger Y, Soultanakis RP, Swisher SG, Uhal BD. Apoptosis in lung pathophysiology. Am J Physiol Lung Cell Mol Physiol 2000;279:L423–L427.[Abstract/Free Full Text]
  15. Tom B, Dendorfer A, Danser AH. Bradykinin, angiotensin-(1–7), and ACE inhibitors: how do they interact? Int J Biochem Cell Biol 2003;35:792–801.[CrossRef][Medline]
  16. Li Y, Sato T. Dual signaling via protein kinase C and phosphatidylinositol 3'-kinase/Akt contributes to bradykinin B2 receptor-induced cardioprotection in guinea pig hearts. J Mol Cell Cardiol 2001;33:2047–2053.[CrossRef][Medline]
  17. Xie P, Browning DD, Hay N, Mackman N, Ye RD. Activation of NF-kappa B by bradykinin through a Galpha(q)- and Gbeta gamma-dependent pathway that involves phosphoinositide 3-kinase and Akt. J Biol Chem 2000;275:24907–24914.[Abstract/Free Full Text]
  18. Houle S, Marceau F. Wortmannin alters the intracellular trafficking of the bradykinin B2 receptor: role of phosphoinositide 3-kinase and Rab5. Biochem J 2003;375:151–158.[CrossRef][Medline]
  19. Graness A, Adomeit A, Heinze R, Wetzker R, Liebmann C. A novel mitogenic signaling pathway of bradykinin in the human colon carcinoma cell line SW-480 involves sequential activation of a Gq/11 protein, phosphatidylinositol 3-kinase beta, and protein kinase Cepsilon. J Biol Chem 1998;273:32016–32022.[Abstract/Free Full Text]
  20. Jin Z, Gao F, Flagg T, Deng X. Nicotine induces multi-site phosphorylation of Bad in association with suppression of apoptosis. J Biol Chem 2004;279:23837–23844.[Abstract/Free Full Text]
  21. Tan Y, Demeter MR, Ruan H, Comb MJ. BAD Ser-155 phosphorylation regulates BAD/Bcl-XL interaction and cell survival. J Biol Chem 2000;275:25865–25869.[Abstract/Free Full Text]
  22. Bergmann A. Survival signaling goes BAD. Dev Cell 2002;3:607–608.[CrossRef][Medline]
  23. Dobbs LG, Gonzalez R, Williams MC. An improved method for isolating type II cells in high yield and purity. Am Rev Respir Dis 1986;134:141–145.[Medline]
  24. Tschumperlin DJ, Margulies SS. Alveolar epithelial surface area-volume relationship in isolated rat lungs. J Appl Physiol 1999;86:2026–2033.[Abstract/Free Full Text]
  25. Winer J, Jung CK, Shackel I, Williams PM. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem 1999;270:41–49.[CrossRef][Medline]
  26. Bao S, Wang Y, Sweeney P, Chaudhuri A, Doseff AI, Marsh CB, Knoell DL. Keratinocyte growth factor induces Akt kinase activity and inhibits Fas-mediated apoptosis in A549 lung epithelial cells. Am J Physiol Lung Cell Mol Physiol 2005;288:L36–42.[Abstract/Free Full Text]
  27. Okano J, Shiota G, Matsumoto K, Yasui S, Kurimasa A, Hisatome I, Steinberg P, Murawaki Y. Hepatocyte growth factor exerts a proliferative effect on oval cells through the PI3K/AKT signaling pathway. Biochem Biophys Res Commun 2003;309:298–304.[CrossRef][Medline]
  28. Oswari J, Matthay MA, Margulies SS. Keratinocyte growth factor reduces alveolar epithelial susceptibility to in vitro mechanical deformation. Am J Physiol Lung Cell Mol Physiol 2001;281:L1068–L1077.[Abstract/Free Full Text]
  29. Wang R, Zagariya A, Ibarra-Sunga O, Gidea C, Ang E, Deshmukh S, Chaudhary G, Baraboutis J, Filippatos G, Uhal BD. Angiotensin II induces apoptosis in human and rat alveolar epithelial cells. Am J Physiol 1999;276:L885–L889.[Medline]
  30. Minshall RD, Tan F, Nakamura F, Rabito SF, Becker RP, Marcic B, Erdos EG. Potentiation of the actions of bradykinin by angiotensin I-converting enzyme inhibitors. The role of expressed human bradykinin B2 receptors and angiotensin I-converting enzyme in CHO cells. Circ Res 1997;81:848–856.[Abstract/Free Full Text]
  31. Benzing T, Fleming I, Blaukat A, Muller-Esterl W, Busse R. Angiotensin-converting enzyme inhibitor ramiprilat interferes with the sequestration of the B2 kinin receptor within the plasma membrane of native endothelial cells. Circulation 1999;99:2034–2040.[Abstract/Free Full Text]
  32. Newton R, Eddleston J, Haddad el B, Hawisa S, Mak J, Lim S, Fox AJ, Donnelly LE, Chung KF. Regulation of kinin receptors in airway epithelial cells by inflammatory cytokines and dexamethasone. Eur J Pharmacol 2002;441:193–202.[CrossRef][Medline]
  33. Kitakaze M, Node K, Takashima S, Minamino T, Kuzuya T, Hori M. Cellular mechanisms of cardioprotection afforded by inhibitors of angiotensin converting enzyme in ischemic hearts: role of bradykinin and nitric oxide. Hypertens Res 2000;23:253–259.[Medline]
  34. Ali MH, Mungai PT, Schumacker PT. Stretch-induced phosphorylation of focal adhesion kinase in endothelial cells: role of mitochondrial oxidants. Am J Physiol Lung Cell Mol Physiol 2006;291:L38–45.[Abstract/Free Full Text]
  35. Hengartner MO. The biochemistry of apoptosis. Nature 2000;407:770–776.[CrossRef][Medline]



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