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Department of Surgery, Texas A&M University System Health Science Center College of Medicine and Scott & White Memorial Hospital, Temple, Texas
Submitted 25 November 2007 ; accepted in final form 7 March 2008
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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m), smac and cytochrome c release, and caspase-3 activity (P < 0.05). In RLMEC monolayers, HS serum and BAK (BH3) peptide both induced hyperpermeability that was inhibited by Ang-1 (P < 0.05). Ang-1 attenuated HS and BAK (BH3) peptide-induced collapse of m, smac release, cytochrome c release, activation of caspase-3, and vascular hyperpermeability. In vivo, BAK (BH3) induced vascular hyperpermeability that was attenuated by Ang-1 (P < 0.05). These findings suggest that Ang-1's role in maintaining microvascular endothelial barrier integrity involves the intrinsic apoptotic signaling cascade.
ischemia-reperfusion injury; microvascular hyperpermeability; smac
Angiopoietin-1 (Ang-1), an endothelial growth factor, is a potent inhibitor of vascular permeability (15, 20, 22, 32, 33). Ang-1 was the first Tie-2 receptor ligand to be identified and was shown to stimulate tyrosine phosphorylation of Tie-2 receptors. Ang-1 has been shown to attenuate human umbilical cord endothelial cell apoptosis by inhibition of smac release from mitochondria (18).
Smac has been shown to facilitate caspase activation by its ability to bind and inhibit the function of the inhibitors of apoptosis proteins (IAPs) (4, 5). Recent studies show that endothelial cell apoptosis can be regulated by inhibition of smac release from mitochondria (18). The release of smac and cytochrome c from the mitochondria results in the activation of caspase-3. Caspase-3 activation has been shown to result in the cleavage of a variety of cell adherens proteins including β-catenin (3, 16). β-Catenin functions as a regulator of VE-cadherin-mediated cell-cell adhesion in endothelial cells, and its cleavage may result in microvascular hyperpermeability. Thus alterations in mitochondrial membrane integrity and the subsequent mitochondrial release of proapoptotic factors such as smac and cytochrome c may have important implications in modulating vascular permeability. While recent studies provide evidence that the mitochondrial release of smac is associated with endothelial cell apoptosis, its relationship to vascular hyperpermeability has not been demonstrated.
We hypothesized that Ang-1 would prevent vascular hyperpermeability following hemorrhagic shock by regulating the intrinsic apoptotic signaling cascade. The purpose of this study was to determine the effect of Ang-1 on vascular hyperpermeability, mitochondrial release of smac and cytochrome c, and caspase-3 activity following hemorrhagic shock in vivo with parallel in vitro studies using microvascular endothelial cells.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Male Sprague-Dawley rats (275–325 g) were obtained from Charles River Laboratories (Wilmington, MA). The rats were housed at our institutional animal facility at 25 ± 2°C, and the humidity was maintained at 55%. They were maintained on a 12:12-h dark-light cycle with free access to food and water. All surgical procedures and experimental protocols described here were conducted at Texas A&M University Health Science Center College of Medicine and Scott and White Hospital after approval by the Institutional Animal Care and Use Committee. The animal facility is approved by the American Association for Accreditation of Laboratory Animal Care in accordance with National Institutes of Health guidelines.
Chemicals and Solutions
The test solute used for the permeability measurements was fluorescein isothiocyanate-bovine albumin (FITC-albumin; Sigma). The test solution was prepared by dissolving the FITC-albumin (50 mg/kg) in saline. Mitochondrial transmembrane potential (m) was determined by using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Cell Technology, Mountain View, CA). JC-1 reagent was prepared by reconstituting the lyophilized reagent with 500 µl of DMSO to obtain a 100x stock solution. Immediately before the experiments the 100x stock solution was diluted 1:100 in 1x assay buffer. Previous studies to determine the effects of DMSO on permeability have been published by our laboratory (8, 10). For in vivo studies, BAK (BH3) peptide (R&D Systems, Minneapolis, MN), 80 µl of a 1 µg/µl stock, was mixed with TransIT (Mirus Bio-Corporation, Madison, WI) for a final concentration of 5 µg/ml and 10 µl/ml (TransIT) of the rat's blood volume. BAK (BH3) peptide at a concentration of 5 µg/ml of culture medium was used to induce hyperpermeability in rat lung microvascular endothelial cell (RLMEC) monolayers. The dose/concentration of Ang-1 (gift from Regeneron Pharmaceuticals, Rensselaer, NY or from R&D Systems; 40 ng/ml for cell culture studies and 200 ng/ml for in vivo studies) and the duration of treatment (10 min before shock) were set based on previously published information on the effect of Ang-1 on Tie-2 receptor tyrosine phosphorylation (18). Rabbit polyclonal antibody for β-catenin and FITC-conjugated anti-rabbit secondary antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The annexin V-FITC staining kit was obtained from Invitrogen.
Animal Surgery and Intravital Microscopy
Male Sprague-Dawley rats (275–325 g) obtained from Charles River Laboratories were housed in the institutional animal facility as described above. Before each experiment the rats were fasted for 18 h and given water ad libitum. The animals were anesthetized with a single intramuscular injection xof 50% urethane (1.5 g/kg). Polyethylene tubing (PE-50, 0.58-mm ID) was placed in the right internal jugular vein to give fluid (normal saline) intravenously at 2 ml/h by continuous infusion pump (Harvard Apparatus, South Natick, MA) and in the right carotid artery for blood withdrawal. Mean arterial pressure (MAP) was monitored continuously with a PE-50 cannula placed in the left femoral artery connected to a blood pressure analyzer (Dig-Med, BPA 400A, Micromed, Louisville, KY). A midline laparotomy incision was performed to expose a section of mesentery from the proximal ileum for exteriorization. The rats were placed in a lateral decubitus position on a temperature-controlled Plexiglas platform mounted to an intravital upright microscope (Nikon E600, Tokyo, Japan). The mesentery was maintained at 37°C. The mesentery was superfused with normal saline at 2 ml/min and covered with plastic wrap to reduce evaporation. Venules with diameters of 20–35 µm were selected for study with a Nikon x20 objective, 0.45- to 2.16-mm working distance (Nikon Instruments, Natick, MA). Images were obtained with a Photometric Cascade Camera (Roper Scientific, Tucson, AZ). A video time and date generator (WJ-810; Panasonic, Secaucus, NJ) provided on-screen time, date, and stopwatch functions. The images were projected onto a computer monitor (Trinitron 20-in. monitor; Sony, New York, NY) and were captured digitally on computer disk. Data were analyzed with MetaMorph 4.5/4.6 (Universal Imaging, Downingtown, PA).
In Vivo Animal Experiments
Effect of angiopoietin-1 on vascular hyperpermeability.
The rats were allowed to recover from surgical manipulation for 30 min before the start of all experiments. This was followed by the recording of baseline parameters: MAP, red blood cell center line velocity, and vessel diameter. During this period, the animals were dosed with FITC-albumin (50 mg/kg) and baseline-integrated optical intensities were obtained intra- and extravascularly (2 sites, same computed areas; mean values were used). The rats were divided into a sham-operated (control) group, a hemorrhagic shock group, a hemorrhagic shock group pretreated with Ang-1 (200 ng/ml), and an Ang-1 alone-treated group. Each experimental group consisted of five rats. The experimental groups of animals then underwent 60 min of hemorrhagic shock. To produce hemorrhagic shock, MAP was decreased to 40 mmHg by withdrawing blood from the right carotid artery into a syringe containing 100 U of heparin. Obtaining this level of shock requires 
40–50% of the animal's blood volume (level IV shock). After the shock period, the shed blood plus two times the volume of normal saline was reinfused to maintain a MAP 
90 mmHg. Parameters were recorded after shock at 10-min intervals for 60 min. Minimal exposures (<15-s recordings) were performed to minimize quenching of the fluorescent indicator.
The extravasation of FITC-albumin was measured by determining the changes in integrated optical intensity by image analysis: I = 1 – (Ii – Io)/Ii, where I is the change in light intensity, Ii is the light intensity inside the vessel, and Io is the light intensity outside the vessel. Each experimental frame was digitized into a 512 x 512 charge-coupled device (CCD) that yielded 16 bits of data/pixel. Grayscale values were measured in the postcapillary venules and in the extravascular space around the venule's per unit area throughout the experiment and at selected times with the MetaMorph image analysis systems. The labeled albumin (FITC) represented relative change in permeability. Areas in the small bowel mesentery, postcapillary venules, and the adjacent extravascular space were selected for study. The images were standardized to images taken at the beginning of each experiment within the same animal and at selected timed intervals between different animals. This method of standardization was selected to minimize the bias incurred with changes in room lighting and hematocrit concentrations.
Effect of angiopoietin-1 on mitochondrial smac protein release. The rats were divided into a sham-operated (control) group, a hemorrhagic shock group, and a hemorrhagic shock group pretreated intravenously with Ang-1 (200 ng/ml). Hemorrhagic shock was induced as described above. The rat mesenteric tissues collected from various experimental groups were homogenized in lysis buffer containing protease inhibitors. Tissue homogenates were centrifuged (12,000 g for 30 min at 4°C) to remove insoluble tissue material. The supernatant was collected and subjected to protein assay [bicinchoninic acid (BCA) method, Pierce]. Samples (50 µg protein) were heated at 95°C for 5 min, subjected to SDS-PAGE electrophoresis, and electrotransferred to nitrocellulose membranes. The membranes were then blocked for 1 h at room temperature with Tris-buffered saline with Tween 20 (TBST) containing 5% milk powder and then incubated overnight at 4°C with primary antibody for smac (goat polyclonal, 1:200; Santa Cruz Biotechnology) and subsequently with horseradish peroxidase-conjugated donkey anti-goat secondary antibody (Santa Cruz Biotechnology) for 1 h. For control, equal amounts (50 µg protein) of the same tissue lysates were loaded separately and immunoblotting was performed as described above. Anti-β-actin (mouse monoclonal; Sigma) was used as the primary antibody. Prestained molecular weight markers (Invitrogen) were used to determine the molecular weight of the proteins. The membranes were later subjected to a chemiluminescence reagent (Pierce) and exposed to X-ray film. Data analyses were performed with Image J software, and data were expressed as a percentage of sham-operated group values.
Effect of angiopoietin-1 on mitochondrial cytochrome c release. The rats were divided into a sham-operated (control) group, a hemorrhagic shock group, a hemorrhagic shock group pretreated with Ang-1 (200 ng/ml), and an Ang-1 alone-treated group. Hemorrhagic shock was induced as described above. Each experimental group consisted of five rats. Hemorrhagic shock was induced in rats as described above and compared to sham-operated animals. Cytosolic cytochrome c levels were estimated with a cytochrome c ELISA kit (MBL, Woburn, MA). Briefly, mesenteric vessels were dissected from the rat, weighed, and homogenized in a cold preparation buffer (10 mM Tris·HCl pH 7.5, 0.3 M sucrose, 10 µM apoptinin, 10 µM pepstatin, 10 µM leupeptin, 1 mM PMSF). The tissue homogenates were centrifuged (10,000 g for 60 min at 4°C), and the supernatant (cytosol fraction) was collected and subjected to protein estimation. The samples were then treated with a conjugate reagent, transferred to microwell strips coated with anti-cytochrome c antibody, and incubated for 60 min at room temperature. The well contents were discarded, and the wells were washed with a wash solution. The samples were then treated with a peroxidase substrate reagent and incubated for 15 min at room temperature. After the addition of a stop solution (0.5 M H2SO4), the optical density of each well was measured at 450 nm within 30 min. A serial dilution of cytochrome c calibrator was subjected to the assay along with the samples, and the values were plotted. The concentration of cytochrome c was calibrated from the standard curve.
Effect of angiopoietin-1 on caspase-3 activity. The rats were divided into a sham-operated (control) group, a hemorrhagic shock group, a hemorrhagic shock group pretreated with Ang-1 (200 ng/ml), and an Ang-1 alone-treated group. Hemorrhagic shock was induced as described above. Each experimental group consisted of five rats. Caspase-3 activity was determined with a caspase-3 activity assay kit (Calbiochem). Briefly, the mesenteric vasculature was collected and homogenized in the sample buffer provided in the assay kit. The tissue lysates were treated with the substrate conjugate and incubated for 2 h at 37°C. The DEVD substrate provided in the assay kit was already labeled with a fluorescent molecule, 7-amino-4-trifluoromethylcoumarin (AFC). The resulting fluorescence was measured in a fluorescent plate reader capable of measuring excitation at 400 nm and emission at 505 nm.
In Vitro Experiments
Effect of angiopoietin-1 on shock serum-induced monolayer hyperpermeability in RLMEC. RLMEC (VEC Technologies, Rensselaer, NY) were maintained on fibronectin-coated dishes in complete MCDB-3 medium supplemented with 10% fetal bovine serum. The cells were later grown on Costar Transwell membranes for 48 h. The cells were treated with serum obtained from sham-operated, shock T0 (60-min shock period followed by 0-min resuscitation), and shock T60 (60-min shock period followed by 60-min resuscitation) rats for 30 min. Hemorrhagic shock was induced as described above. The cells were exposed to Ang-1 (40 ng/ml of phenol red-free cell culture medium) 15 min before the exposure to serum. Untreated (basal) and Ang-1 alone-treated groups served as controls. FITC-albumin (5 mg/ml final concentration) was added to the luminal chamber and left for 30 min. The samples (100 µl) collected from the abluminal (lower) chambers were analyzed for FITC fluorescent intensity with a fluorometric plate reader at excitation of 494 nm and 520 nm. The data were calculated as percentage of the control (basal) values.
Effect of angiopoietin-1 on BAK (BH3)-induced monolayer hyperpermeability in RLMEC. RLMEC were grown on Costar Transwell membranes as described above. The cells were transfected with BAK (BH3) peptide (5 µg/ml final concentration). The transfection medium (TransIT-LT1 polyamine) was exposed to BAK (BH3) peptide (5 µg/ml) for 15 min, and the transfection was performed for 1 h. The cells were exposed to Ang-1 (40 ng/ml) 15 min before BAK (BH3) transfection. Untreated (basal), Ang-1 alone-treated, and TransIT alone-treated cells served as controls. FITC-albumin (5 mg/ml final concentration) was added to the luminal chamber for 30 min. The samples (100 µl) collected from the abluminal (lower) chambers were analyzed for FITC fluorescent intensity with a fluorometric plate reader at excitation of 494 and 520 nm. The data were calculated as percentage of the control (basal) values.
Effect of angiopoietin-1 on BAK (BH3)-induced collapse of mitochondrial membrane potential in RLMEC. To determine the mitochondrial membrane potential, RLMEC were grown on fibronectin-coated glass coverslips for 24 h and exposed to medium without phenol red for 1 h. The transfection medium (TransIT-LT1 polyamine) was exposed to BAK (BH3) peptide (5 µg/ml) for 15 min before transfection for 1 h, as described above. The cells were exposed to Ang-1 (40 ng/ml) 15 min before BAK (BH3) transfection. Untreated, Ang-1 alone-treated, and TransIT alone-treated cells served as controls. The cells were incubated with JC-1 for 15 min at 37°C, washed in PBS, and observed immediately under a Leica AOBS SP2 confocal microscope. JC-1 aggregates were viewed with a band-pass filter designed to detect Texas red (excitation 590 nm, emission 610 nm). JC-1 monomers were detected with a band-pass filter used for the detection of fluorescein (excitation 490 nm, emission 520 nm).
Effect of angiopoietin-1 on BAK (BH3)-induced smac release in RLMEC. To determine smac protein expression, cells were grown on fibronectin-coated dishes for 24 h. The transfection medium (TransIT-LT1 polyamine) was exposed to BAK (BH3) peptide (5 µg/ml) for 15 min before transfection for 1 h, as described above. The cells were exposed to Ang-1 (40 ng/ml) 15 min before BAK (BH3) transfection. Untreated cells served as controls. Cytosolic smac expression was determined by immunoblotting. The cell lysates were centrifuged (12,000 g for 30 min at 4°C), and the supernatant was collected and subjected to protein assay (BCA method, Pierce). The protein samples were heated at 95°C for 5 min, subjected to SDS-PAGE electrophoresis, and electrotransferred to nitrocellulose membranes, as described above. The membranes were then blocked for 1 h at room temperature with TBST containing 5% milk powder and incubated overnight at 4°C with primary antibody for smac (goat polyclonal, 1:200; Santa Cruz Biotechnology) and subsequently with horseradish peroxidase-conjugated donkey anti-goat secondary antibody (Santa Cruz Biotechnology) for 1 h. The membranes were later subjected to a chemiluminescence reagent (Pierce) and exposed to X-ray film. Mouse monoclonal antibody for β-actin (Sigma) was used as an internal control. Prestained molecular weight markers (Invitrogen) were used to determine the molecular weight of the proteins. Four replicates from each group were used for the immunoblot analysis.
Effect of angiopoietin-1 on BAK (BH3)-induced cytochrome c release in RLMEC. RLMEC were grown on fibronectin-coated culture dishes at 37°C with 5% CO2 as described above. The transfection medium (TransIT) was exposed to BAK (BH3) peptide (5 µg/ml) for 15 min, and the transfection was performed for 1 h. The cells were exposed to Ang-1 (40 ng/ml) 15 min before BAK (BH3) transfection. Untreated, Ang-1 alone-treated, and TransIT alone-treated cells served as controls. Cytosolic cytochrome c levels were estimated with a cytochrome c ELISA kit. Briefly, the cells were lysed in a cold preparation buffer provided in the kit and centrifuged (10,000 g for 60 min at 4°C), and the supernatant (cytosolic fraction) was collected and subjected to protein assay (BCA method, Pierce). The samples were treated with a conjugate reagent, transferred to a cytochrome c antibody-coated microwell plate, and incubated at room temperature for 60 min. The wells were washed and treated with a substrate reagent and incubated for 30 min, followed by addition of a stop solution. The optical density was read at 450 nm in a calorimetric plate reader. A serial dilution of cytochrome c calibrator was subjected to the assay along with the samples, and the values were plotted. The concentration of cytochrome c was calibrated from the standard curve.
Effect of angiopoietin-1 on caspase-3 activity in RLMEC. The transfection medium (TransIT) was exposed to BAK (BH3) peptide (5 µg/ml) for 15 min, and the transfection was performed for 1 h. The cells were exposed to Ang-1 (40 ng/ml) 15 min before BAK (BH3) transfection. Untreated, Ang-1 alone-treated, and TransIT alone-treated cells served as controls. Caspase-3 activity was determined with a caspase-3 activity assay kit (Calbiochem) as described above. The DEVD substrate provided in the kit was labeled with a fluorescent molecule, AFC. The cell lysate was used for protein estimation and treated with the substrate conjugate. The resulting fluorescent intensity was measured in a fluorescent plate reader capable of measuring excitation at 400 nm and emission at 505 nm.
Effect of hemorrhagic shock serum on endothelial adherens junction and annexin V binding. RLMEC were grown on fibronectin-coated glass chamber slides. The cells were treated with serum obtained from sham-operated or shock T60 rats for 30 min. A control group of cells received no treatment. Hemorrhagic shock was induced as described above. After the serum treatment, cells were washed in PBS and fixed in 4% paraformaldehyde. After repeated washing steps, Triton X-100 treatment, and blocking for nonspecific binding, cells were incubated with a primary antibody for β-catenin (1:100) raised in rabbit (Santa Cruz Biotechnology) at 4°C overnight. Cells were washed in PBS and exposed to an FITC-conjugated secondary antibody for 1 h. After repeated washing steps, the cells were mounted in an antifade mounting medium that contained the nuclear stain DAPI (Vectashield) and observed under a fluorescent microscope with appropriate filters for visualizing FITC and DAPI.
Annexin V is a phospholipid binding protein with high affinity for phosphatidylserine found in the outer cell membrane beginning early in the process of apoptosis. During apoptosis, a redistribution of phosphatidylserine from the cytoplasmic location to the outer leaflet takes place, making it available for annexin V binding. For the annexin V-FITC binding study, control cells and serum-treated cells were exposed to 5 µl of annexin V-FITC (Invitrogen) per chamber, incubated for 15 min in the dark, and washed in PBS, followed by exposure to annexin V binding buffer. The cells were observed under a fluorescent microscope to visualize annexin V-FITC fluorescence.
In Vivo BAK Transfection: Effect of Angiopoietin-1 on BAK-Induced Vascular Hyperpermeability
BAK (BH3) peptide transfection in rats was previously shown to induce hyperpermeability similar to that of hemorrhagic shock in rats (7). This experiment was designed to test whether Ang-1 prevents BAK (BH3)-induced vascular hyperpermeability. The rats were divided into a sham-operated group, a transfection reagent (TransIT)-treated group, a BAK (BH3) peptide (5 µg/ml blood volume)-treated group, and a BAK (BH3) peptide (5 µg/ml blood volume)-group pretreated with Ang-1 (200 ng/ml). Each experimental group consisted of five rats. BAK (BH3) peptide solution was prepared by exposing the peptide to a transfection reagent (TransIT) as described previously (7). The extravasation of FITC-albumin to determine vascular hyperpermeability was measured as described above.
Statistical Analysis
All data are expressed as means ± SE. Student's t-test and analysis of variance (ANOVA) were used for determining significant differences between two mean values. Intergroup comparisons were made with ANOVA followed by the Bonferroni posttest for multiple comparisons. In in vivo vascular permeability studies, each experimental value was compared to initial baseline value and expressed as percent change. This method decreases bias between animals due to red blood cell accumulation and changes in room lighting. A P value of <0.05 was considered to indicate a statistically significant difference.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Hemorrhagic shock induced hyperpermeability in rat mesenteric postcapillary venules, which was attenuated by Ang-1 pretreatment. Figure 1A is a composite image of a rat mesenteric postcapillary venule. In control, minimal extravasation of FITC-albumin into the extravascular space was observed. The second image in Fig. 1A corresponds to 60 min of hemorrhagic shock and 60 min of resuscitation (T60 group). Hemorrhagic shock resulted in a marked increase in FITC-albumin extravasation. The third image in Fig. 1A corresponds to Ang-1 treatment before 60 min of hemorrhagic shock and 60 min of resuscitation.
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Angiopoietin-1 Inhibits Hemorrhagic Shock-Induced smac Release
The release of smac from mitochondria to the cytosol occurs after the opening of the mitochondrial transition pores. Opening of mitochondrial transition pores is directly related to the decrease in m. Release of smac from mitochondria to the cytosol inhibits the functions of IAPs, leading to the activation of caspase-3. Figure 2 shows representative immunoblot images (Fig. 2A) and corresponding quantitative data (Fig. 2B) for smac protein obtained from the sham-operated group, the shock group, and the shock group pretreated with Ang-1. In rat mesenteric tissue collected from sham-operated animals, cytosolic smac levels were low. Hemorrhagic shock for 1 h followed by 60 min of resuscitation (T60 group) resulted in an increase in cytosolic smac compared with the sham-operated group (185.72 ± 8.5% of sham-operated group values; P < 0.05). Ang-1 treatment before hemorrhagic shock and 60 min of resuscitation (T60 group) resulted in significant decrease in smac levels compared with the hemorrhagic shock group (102.05 ± 3.7% of sham-operated group values; P < 0.05). These results show that Ang-1 attenuated hemorrhagic shock-induced release of mitochondrial smac, an activator of caspase-3.
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The release of cytochrome c from mitochondria to the cytosol occurs after the opening of the mitochondrial transition pores. Cytochrome c release to the cytosol and subsequent activation of caspase-3 has been reported to be the key event in apoptosis induced by various stimuli. Cytosolic cytochrome c levels were elevated in the T60 group (43.98 ± 5.39 ng/mg protein) compared with the sham-operated group (18.03 ± 42.5 ng/mg protein) (Fig. 3A; P < 0.05). Cytochrome c levels in the hemorrhagic shock group pretreated with Ang-1 (40 ng/ml) showed a significant decrease (25.51 ± 2.7 ng/mg protein) compared with the hemorrhagic shock group (Fig. 3A; P < 0.05). The Ang-1 alone-treated group showed no significant change in cytochrome c levels (21.61 ± 42.5 ng/mg protein) compared with the sham-operated group. These results show that Ang-1 attenuated hemorrhagic shock-induced release of cytochrome c from mitochondria to the cytosol.
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Caspase-3 activation occurs after cytochrome c release from mitochondria. Caspase-3 activation leads to the proteolytic cleavage of a variety of cellular substrates including endothelial cell adherens junction proteins. The caspase-3 activity assay has been used to detect caspase-3 activation in tissue samples and cells. Hemorrhagic shock induced caspase-3 activity in rat mesenteric tissue versus sham-operated group values (205 ± 12.38%, Fig. 3B; P < 0.05). Rats pretreated with Ang-1 followed by hemorrhagic shock showed significant decrease in caspase-3 activity compared with the hemorrhagic shock group (108.63 ± 19.26%, Fig. 3B; P < 0.05). Rats treated with Ang-1 followed by hemorrhagic shock were similar to the sham-operated animals. The Ang-1 alone-treated group showed no significant change in caspase-3 activity (93.39 ± 17.94%) compared with the sham-operated control group. These results indicate that Ang-1 prevented hemorrhagic shock-induced caspase-3 activation. This might prevent caspase-3 mediated proteolytic cleavage of endothelial cell adherens proteins such as β-catenins.
Angiopoietin-1 Attenuates Hemorrhagic Shock Serum-Induced Monolayer Hyperpermeability in RLMEC
Serum from sham-operated animals did not induce RLMEC monolayer hyperpermeability (102.3 ± 6.2%; Fig. 4A). Serum obtained from rats after 60 min of hemorrhagic shock and 0 min of resuscitation (T0 group) or 60 min of resuscitation (T60 group) induced hyperpermeability (172 ± 15.9% and 165 ± 9.3%, respectively, Fig. 4A; P < 0.05). Ang-1 (40 ng/ml) pretreatment significantly attenuated the hyperpermeability (115 ± 9.7%; P < 0.05, Fig. 4A). Ang-1 treatment alone did not show any significant change in permeability compared with untreated (basal) or sham serum-treated cells (112.3 ± 14% of basal).
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BAK (BH3) peptide transfection significantly increased permeability versus untreated (basal) cells (226 ± 16%, Fig. 4B; P < 0.05). Pretreatment with Ang-1 decreased the BAK-induced hyperpermeability (115 ± 19%, Fig. 4B; P < 0.05). Treatment with Ang-1 alone showed no significant change in permeability compared with untreated (basal) cells (118 ± 18%). Treatment with TransIT alone also showed no significant change in permeability compared with untreated (basal) cells (data not shown).
Angiopoietin-1 Prevents BAK-Induced Collapse Of Mitochondrial Transmembrane Potential in RLMEC
BAK (BH3)-transfected cells showed a decrease in red fluorescence (J aggregates) compared with control cells, indicating the collapse of m (Fig. 5). BAK (BH3)-transfected cells pretreated with Ang-1 showed no visible change in red fluorescence compared with the control cells, indicating intact mitochondria. In nonapoptotic cells, JC-1 exists as a monomer in the cytosol (green) and also accumulates as J aggregates in the mitochondria, which fluoresce as red. On induction of apoptosis and mitochondrial degradation, JC-1 exists only in a monomeric form (green). Ang-1 alone- or TransIT alone-treated cells showed no change in mitochondrial membrane potential.
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In untreated RLMEC, smac cytosolic content was very low or not detected (Fig. 6A). BAK (BH3)-transfected cells showed increase in smac cytosolic content compared with untreated cells (Fig. 6A). BAK (BH3)-transfected cells pretreated with Ang-1 demonstrated significant attenuation of smac release evident from the decrease in smac cytosolic content.
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In RLMEC, the cytosolic levels of cytochrome c were significantly higher (47.1 ± 5.1 ng/mg protein) after BAK (BH3) transfection compared with the control cells (28.6 ± 5.48 ng/mg protein; P < 0.05; Fig. 6B). Cytosolic cytochrome c levels were significantly attenuated in BAK (BH3)-transfected cells (30.13 ± 5.03 ng/mg protein; P < 0.05; Fig. 6B) pretreated with Ang-1 (40 ng/ml) compared with BAK (BH3)-transfected cells without Ang-1 treatment (26.7 ± 7.1 ng/mg protein; P < 0.05; Fig. 6B). No significant difference in cytochrome c levels was observed in TransIT alone-treated cells (26.6 ± 4.5 ng/mg protein) compared with untreated control cells.
Angiopoietin-1 Inhibits BAK-Induced Caspase-3 Activation in RLMEC
In BAK (BH3)-transfected cells, caspase-3 activity was significantly higher compared with control cells (208.3 ± 5.9%; P < 0.05; Fig. 7). Caspase-3 activity was significantly decreased (127.6 ± 4.6%) in BAK (BH3)-transfected cells pretreated with Ang-1 compared with BAK (BH3)-transfected cells without Ang-1 treatment (P < 0.05; Fig. 7). BAK (BH3)-transfected cells treated with Ang-1 showed no significant difference in caspase-3 activity compared with control cells (P < 0.05; Fig. 7). No significant difference in caspase-3 activity was observed between TransIT alone-treated cells and untreated control cells.
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Control cells showed strong and continuous β-catenin immunofluorescence at the endothelial cell-cell junctions (Fig. 8), indicating an intact cell barrier. Treatment with serum obtained from rats after 60 min of hemorrhagic shock and 60 min of resuscitation (T60 group) induced disruption of endothelial cell junctions evidenced by irregular and scattered β-catenin fluorescence (Fig. 8), indicating disruption of adherens junctions. Serum from sham-operated animals did not induce disruption of adherens junctions.
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Angiopoietin-1 Decreases BAK (BH3)-Induced Vascular Hyperpermeability in Vivo
Figure 9A shows representative images of rat mesenteric postcapillary venules from control, BAK (BH3) transfection, and Ang-1 treatment before BAK (BH3) transfection. Control images show minimum FITC-albumin extravasation, whereas BAK (BH3) transfection shows increased extravasation indicating hyperpermeability. Ang-1 pretreatment shows minimum FITC-albumin extravasation, indicating the protective effects of Ang-1. Figure 9B shows a graphic representation of results from this experiment. Mesenteric postcapillary venules of rats transfected with BAK (BH3) peptide showed hyperpermeability versus sham- or TransIT alone-treated rats (P < 0.05). Rats pretreated with Ang-1 followed by BAK (BH3) transfection showed a significant decrease in permeability compared with BAK (BH3) peptide-transfected rats without Ang-1 pretreatment (P < 0.05). Rats transfected with BAK (BH3) after Ang-1 showed no significant change in permeability versus the sham-treated group or the TransIT alone-treated group (P < 0.05).
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DISCUSSION |
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m resulted in the release of smac and cytochrome c into the cytosol. Ang-1 pretreatment prevented the smac and cytochrome c release into the cytoplasm, suggesting its protective functions regulating mitochondrial membrane potential. In parallel studies conducted in RLMEC, serum collected after hemorrhagic shock and tested on microvascular endothelial cells disrupted the adherens junctions. Ang-1 attenuated monolayer hyperpermeability. The monolayer permeability was induced either by serum from hemorrhagic shock rats or by BAK (BH3) peptide transfection. Previous studies from our laboratory (7) demonstrated that the proapoptotic factor BAK is a mediator of vascular hyperpermeability following hemorrhagic shock. A synthetic BAK (BH3) peptide was shown to induce vascular hyperpermeability after hemorrhagic shock, whereas a mutant BAK (BH3) peptide was ineffective (7). In addition to the observations in vivo, our results show that Ang-1 treatment prevented BAK (BH3)-induced collapse of m and release of smac and cytochrome c and also inhibited BAK (BH3)-induced caspase-3 activation in RLMEC. These observations suggest that the treatment with Ang-1 effectively controlled vascular hyperpermeability in hemorrhagic shock by regulating smac- and cytochrome c-mediated intrinsic apoptotic signaling. Hemorrhagic shock is known to induce vascular hyperpermeability (8–11, 25). We recently showed (7) the involvement of intrinsic apoptotic signaling in vascular hyperpermeability following hemorrhagic shock. In human umbilical cord endothelial cells, induction of apoptosis by serum deprivation for 6 h increased cytoplasmic cytochrome c and smac protein levels (18). Our study provides further evidence of the involvement of proapoptotic factors in vascular hyperpermeability and also shows how the selective regulation of various steps in the process is achieved by using an inhibitor of vascular permeability. Our results show that hemorrhagic shock stimulated the release of the proapoptotic factor smac from mitochondria (evident from the increase in smac levels in the cytoplasm) in the rat mesenteric vasculature. The release of the mitochondrial protein smac was previously shown to induce caspase activation through its ability to bind to and inhibit the function of members of the IAP family (5, 24, 26). Thus our observations support the involvement of a caspase-dependent apoptotic pathway in vascular permeability following hemorrhagic shock.
Hemorrhagic shock-induced collapse of mitochondrial permeability transition may have resulted in the mitochondrial release of smac and cytochrome c to the cytosol. Although the precise mitochondrial mechanisms that mediate hemorrhagic shock-mediated smac release are not known, mitochondrial reactive oxygen species (ROS) may have a significant role in this. ROS-induced oxidative stress is one of the most important stimulants of apoptotic cell death (23). Oxidative stress is known to alter mitochondrial permeability transition (38). Mitochondrial transition pores are major targets of ROS, and ROS have been implicated in vascular hyperpermeability following hemorrhagic shock in rats (9, 10, 38). Mitochondrial oxidative stress caused by ROS after reperfusion injury leads to accumulation of cytosolic smac and release of cytochrome c (30, 31). Hydrogen peroxide-induced apoptosis in cardiomyocytes and myogenic cells was accompanied by the release of smac from mitochondria, activation of caspase-9 and caspase-3, and DNA fragmentation (21). It is quite possible that in hemorrhagic shock-induced vascular hyperpermeability mitochondrial oxidative stress played an important role in the translocation of smac and cytochrome c to the cytoplasm through mitochondrial transition pores. The preservation of mitochondrial transition pores by Ang-1 is thus an effective approach to control mitochondrial release of smac and cytochrome c to the cytosol and to prevent vascular hyperpermeability.
Ang-1 has antiapoptotic effects in endothelial cells and protective function against vascular leakage (18, 32). The angiopoietin/Tie receptor system is known to contribute to angiogenesis and vascular remodeling by mediating interactions of endothelial cells with smooth muscle cells and pericytes (24). Ang-1-overexpressing mice have leakage-resistant blood vessels, and Ang-1 protected adult vasculature from leaking, counteracting the potentially lethal actions of VEGF and inflammatory agents (32, 33). Further studies showed that Ang-1 inhibited mitochondrial smac release and caspase-3 activity in human umbilical vein endothelial cells subjected to apoptotic signaling (18). Our results showing that Ang-1 inhibited shock serum-induced as well as proapoptotic factor BAK-induced microvascular hyperpermeability suggest that Ang-1 exerts its protective effects on hyperpermeability in part through its inhibitory actions on the intrinsic apoptotic signaling pathway.
The cellular and molecular mechanisms by which Ang-1 protected mitochondrial membrane integrity are not clearly understood at this time. Ang-1 is known to attenuate apoptotic cell death via phosphatidylinositol 3-kinase/AKT-dependent pathway and inhibition of smac release from the mitochondria (18, 19, 35). It is possible that Ang-1, in addition to its direct protective effects on mitochondrial transition pores, targeted and inhibited proapoptotic proteins that induce smac release from mitochondria. Activation of the mitochondrial pathway with the activation of the effector caspase-3 plays an important role in endothelial cell apoptosis (1, 18, 34). Smac is released from mitochondria to the cytoplasm and is known to potentiate the functions of cytochrome c-apoptosome complex (14). It has been demonstrated that death receptor-initiated mitochondrial release of cytochrome c and smac is caspase dependent (21). Smac promotes not only the proteolytic activation of procaspase-3 but also the enzymatic activity of mature caspase-3, both of which are dependent on its ability to interact physically with the IAPs (18, 19). The IAP family inhibits apoptosis by binding the catalytic domains of caspases-3, -7, and -9 (13, 19). Smac binds to one of the BIR domains of IAPs and blocks the caspase binding sites on IAPs. PR39, a proline- and arginine-rich peptide implicated in myocardial ischemia protection, inhibited hypoxia-induced apoptosis and decreased caspase-3 activity through an increase of IAP-2 expression in endothelial cells (37). Similarly, decreased expression of antiapoptotic proteins Bcl-2 and c-IAP-1 resulting in the activation of apoptotic signaling pathway, involving release of cytochrome c and smac and activation of caspase-9 and then caspase-3, has been observed in human coronary artery endothelial cells (6). Similarly, transgenic mice overexpressing X-linked inhibitor of apoptosis (XIAP) showed protection against hypoxia-ischemia (36). Our finding of the inhibition of smac release and caspase-3 activation by Ang-1 suggests its permissive role on XIAP.
Our results show that disruption of β-catenin/endothelial cell adherens junction occurs after treatment of cells with hemorrhagic shock serum. However, hemorrhagic shock serum does not induce phosphatidylserine externalization, an indicator of cell membrane damage normally observed in apoptotic cells. This study supports our hypothesis that endothelial cell hyperpermeability occurs because of activation of caspase-3 and subsequent cleavage of β-catenin resulting in cell-cell detachment and may not be due to apoptotic cell death. Caspase-3 cleaves a variety of cell adhesion proteins. The caspase-3-dependent cleavage of β-catenin occurs during apoptosis (3, 16, 29). In endothelial cells, β-catenin functions as a regulator of cadherin-mediated cell-cell adhesion. Thus Ang-1-mediated maintenance of m and inhibition of smac and cytochrome c release may be possible mechanisms by which it prevents caspase-3 activation and protects endothelial cell barrier integrity to prevent vascular hyperpermeability.
In this study we utilized an in vivo mesenteric postcapillary venular preparation to study changes in microvascular hyperpermeability. In addition, parallel studies were performed in RLMEC monolayers. Endothelial cells from the lung and mesenteric venules were of particular interest because of the associated morbidity observed secondary to pulmonary edema and intra-abdominal compartment syndrome. It also allowed us to study two different vascular systems to determine whether activation of the apoptotic cascade has similar effects on barrier integrity.
In conclusion, our findings suggest that hemorrhagic shock induces mitochondrial release of proapoptotic smac and cytochrome c. The mitochondrial release of smac and cytochrome c may subsequently activate caspase-3, leading to the disruption of cell adherens junctions and vascular hyperpermeability. Ang-1 treatment maintains mitochondrial membrane integrity, prevents mitochondrial smac and cytochrome c release and caspase-3 activation, and attenuates vascular hyperpermeability. The Ang-1-mediated protective effects may be due to its inhibitory effects on caspase-mediated cleavage of endothelial cell adherens junction proteins and disruption of cell-cell junctions. Ang-1 could be tested as a therapeutic protein for specific protection against hemorrhagic shock-induced endothelial cell injury and vascular leakage.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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