Felicia A. Hunter

In vivo visualization of reactive oxidants and leukocyte-endothelial adherence following hemorrhagic shock

The generation of oxygen radicals during leukocyte-endothelial cell interaction is considered to represent one of the fundamental steps of microvascular injury following ischemia and reperfusion. Indirect evidence also suggests that this relationship may be important following hemorrhagic shock. The purpose of this study was to characterize the temporal changes of reactive oxygen species (ROS) in the mesenteric microvascular endothelium, in vivo, as a consequence of hemorrhagic shock and reperfusion, and to correlate this ROS production to leukocyte adherence. Following a control period, blood was withdrawn to reduce the mean arterial pressure to 40 mmHg for 1 h in urethane-anesthetized rats. Mesenteric venules in a transilluminated segment of small intestine were examined to quantitate changes in ROS generation and leukocyte adherence. Sprague-Dawley rats were injected with dihydrorhodamine 123, a hydroperoxide-sensitive fluorescent probe that is trapped within viable cells as a nonfluorescent form and then converted to the mitochondrion-selective form rhodamine 123 by hydroperoxides. The fluorescent light emission from rhodamine 123 was recorded with digital microscopy and downloaded to a computerized image analysis program. Our results demonstrated an 80% increase in ROS generation beginning within 5 min into resuscitation and a 10-fold increase in leukocyte adherence that occurred at 10 min after resuscitation. Both ROS generation and leukocyte adherence were attenuated with pre-shock administration of platelet activating factor (PAF) antagonist, WEB 2086, and the CD11/CD18a antibody, anti-LFA-1&bgr;. Our findings suggest that ROS production in endothelial cells is increased during reperfusion following hemorrhagic shock and that the mechanism of expression is mediated in part by both PAF expression and subsequent leukocyte adherence.

Angiopoietin-1 inhibits intrinsic apoptotic signaling and vascular hyperpermeability following hemorrhagic shock.

Studies from our laboratory demonstrated the involvement of intrinsic apoptotic signaling in hyperpermeability following hemorrhagic shock (HS). Angiopoietin 1 (Ang-1), a potent inhibitor of hyperpermeability, was recently shown to inhibit apoptosis. The purpose of our study was to determine the effectiveness of Ang-1 in attenuating HS-induced hyperpermeability and its relationship to apoptotic signaling. HS was induced in rats by withdrawing blood to reduce the mean arterial pressure to 40 mmHg for 1 h, followed by reperfusion. Mesenteric postcapillary venules were examined for changes in hyperpermeability by intravital microscopy. Mitochondrial release of second mitochondrial derived activator of caspases (smac) and cytochrome c were determined by Western blot and ELISA, respectively. Caspase-3 activity was determined by fluorometric assay. Parallel studies were performed in rat lung microvascular endothelial cell (RLMEC) monolayers, utilizing HS serum and the proapoptotic Bcl-2 homologous antagonist/killer [BAK (BH3)] peptide as inducers of hyperpermeability. In rats, Ang-1 (200 ng/ml) attenuated HS-induced hyperpermeability versus the HS group (P < 0.05). Ang-1 prevented HS-induced collapse of mitochondrial transmembrane potential (DeltaPsi(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 DeltaPsi(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-1s role in maintaining microvascular endothelial barrier integrity involves the intrinsic apoptotic signaling cascade.

Hypothermia reduces microvascular permeability and reactive oxygen species expression after hemorrhagic shock

BACKGROUND Hypothermia is a frequent manifestation after trauma-induced hemorrhagic shock. Clinical studies have suggested that hypothermia is an independent risk variable predisposing patients to an increase in morbidity. Thus, most of the current goal-directed resuscitation protocols are aimed at the establishment of euthermia. However, recent data suggest that hypothermia may provide protection by attenuating the inflammatory response after hemorrhagic shock. The purpose of this study was twofold: to examine the effects of mild to moderate hypothermia on barrier function after hemorrhagic shock, and to determine the role of reactive oxygen species (ROS) in this process. METHODS After a control period, blood was withdrawn to reduce the mean arterial pressure to 40 mm Hg for 1 hour in urethane-anesthetized rats. Mesenteric postcapillary venules in a transilluminated segment of small intestine were examined to quantitate changes in permeability and ROS expression. Sprague-Dawley rats received an intravenous injection of fluorescein isothiocyanate (FITC)-albumin during the control period. The fluorescent light intensity emitted from the FITC-albumin was recorded with digital microscopy within the lumen of the microvasculature and compared with the intensity of light in the extravascular space. The images were downloaded to a computerized image analysis program that quantitates changes in light intensity. This change in light intensity represents albumin-FITC extravasation. RESULTS Our results demonstrated a marked increase in albumin leakage after hemorrhagic shock that was significantly attenuated with mild (34 degrees C) and moderate (30 degrees C) hypothermia. In addition, hypothermia attenuated ROS expression after hemorrhagic shock. CONCLUSION These data suggest that hypothermia may protect barrier integrity after hemorrhagic shock by inhibition of oxygen radical expression.

Leukocyte adherence and sequestration following hemorrhagic shock and total ischemia in rats

The pathogenesis of generalized microvascular injury following hemorrhagic shock and total ischemia appears to be dependent on leukocytes interacting with the venular endothelium. The purpose of this study was to compare leukocyte adherence and sequestration following hemorrhagic shock with that of total ischemia in the small bowel mesentery of rats. Leukocyte adherence and sequestration was measured by direct visualization in vivo using intravital microscopy. In addition, sequestration was also quantitated by measuring tissue levels of myeloperoxidase, a marker of leukocyte infiltration. Mean arterial blood pressure was decreased to 40 mm Hg for 30 min (hemorrhagic shock group). In the total ischemia group, both the superior and inferior mesenteric arteries were clamped for 30 min followed by reperfusion. Hemorrhagic shock (9.4+/-1.5 cell/100 microm) and total ischemia (8.3+/-3 cell/100 microm) caused a statistically significant increases in leukocyte adherence 60 min postinsult as compared with controls (.9+/-1.5 cell/100 microm). However, the increase in leukocyte adherence appeared earlier and to a greater degree initially following total ischemia. Leukocyte sequestration as measured by intravital microscopy was significant only after total ischemia [(24.6+/-1.7 cell/(100 microm)2; p<.01] and not hemorrhagic shock [3.4+/-.6 cell/(100 microm)2] versus controls [2.2+/-.2 cell/(100 microm)2]. This difference in sequestration was also confirmed by tissue levels of myeloperoxidase. The results of this study suggest that the microvascular response following hemorrhagic shock is different than that of total ischemia, and caution is warranted when extrapolating the experimental results of one to the other.

Vascular Leak in a Rat Model of Preeclampsia

Background/Aims: Preeclampsia is a hypertensive disorder which develops de novo in women during pregnancy. The urinary excretion of the cardiotonic steroid, marinobufagenin (MBG), is increased prior to the development of hypertension. Preeclamptic patients are volume expanded but much of the excess salt and water appears to be located primarily in the interstitial space. Therefore, ‘capillary leak’ syndrome has been postulated in this disorder. Methods: We evaluated the vascular leakage in normal rats following MBG injection and in a rat model of human preeclampsia. We measured the changes in light intensity comparing that in the intravascular to the extravascular space by assessing ‘leak’ of fluorescein-labeled albumin (FITC-albumin) from mesenteric postcapillary venules. Results: FITC-albumin extravasation continued to increase in a time-dependent fashion after MBG infusion and was significant (p < 0.05) at 60 min of observation when compared to sham rats. We also observed a significant difference in ‘vascular leakage’ in preeclamptic rats compared to control non-pregnant and normal pregnant groups starting at 20 min after the FITC-albumin infusion. Conclusion: We propose that MBG is involved in the production of a ‘vascular leak’ in our rat model of preeclampsia.

17β-estradiol Mediated Protection Against Vascular Leak After Hemorrhagic Shock: Role Of Estrogen Receptors And Apoptotic Signaling

Vascular hyperpermeability is a clinical complication associated with hemorrhagic shock (HS) and occurs mainly because of the disruption of the adherens junctional complex. The objective of this study was to understand the role of 17&bgr;-estradiol in HS-induced hyperpermeability particularly focusing on estrogen receptors. In male Sprague-Dawley rats, HS was induced by withdrawing blood to reduce the mean arterial pressure to 40 mmHg for 1 hour followed by 1 hour of resuscitation to 90 mmHg. The study groups were 17&bgr;-estradiol, tamoxifen, fulvestrant plus 17&bgr;-estradiol, propyl pyrazole triol plus 17&bgr;-estradiol, and diarylpropionitrile plus 17&bgr;-estradiol. Intravital microscopy was used to study changes in mesenteric postcapillary venules. Mitochondrial reactive oxygen species formation was studied in vivo using dihydrorhodamine 123. The mitochondrial transmembrane potential was studied using the fluorescent cationic probe 5,5&vprime;,6,6&vprime;tetrachloro-1,1&vprime;,3,3&vprime;tetraethylbenzimidazolyl carbocyanine iodide (JC-1). The mesenteric microvasculature was analyzed for cytochrome c levels by enzyme-linked immunosorbent assay and caspase-3 activity by a fluorometric assay. Our results demonstrated that 17&bgr;-estradiol attenuated HS-induced hyperpermeability. Fulvestrant reversed this protective effect (P < 0.05). Tamoxifen 5 mg/kg attenuated HS-induced hyperpermeability, whereas 10 mg/kg induced permeability (P < 0.05). Both &agr; and &bgr; estrogen receptor agonists inhibited HS-induced hyperpermeability (P < 0.05). 17&bgr;-Estradiol decreased HS-induced reactive oxygen species formation and restored mitochondrial transmembrane potential. 17&bgr;-Estradiol decreased both cytosolic cytochrome c level and activation of caspase-3 (P < 0.05). These findings suggest that 17&bgr;-estradiol protects the microvasculature after HS, and that this protection may be mediated through the &agr; and &bgr; estrogen receptors.

Cyclosporine A Prevents Vascular Hyperpermeability After Hemorrhagic Shock by Inhibiting Apoptotic Signaling

BACKGROUND Hemorrhagic shock (HS) is associated with the activation of caspase-dependent or -independent apoptotic signaling pathways, disruption of endothelial cell adherens junctions, and vascular hyperpermeability. Recent studies have suggested that the vascular hyperpermeability observed after HS is associated with activation of the intrinsic apoptotic signaling cascade resulting in caspase-mediated cleavage of endothelial cell adherens proteins and subsequent cell-cell detachment. We hypothesized that cyclosporine A (CsA) would attenuate vascular hyperpermeability after HS by protecting mitochondrial transition pores and thereby preventing the activation of caspase-mediated apoptotic signaling. The objective of this study was to determine the effect of CsA on, HS-induced hyperpermeability, mitochondrial membrane depolarization, mitochondrial release of cytochrome c, and caspase 3 activation. METHODS HS was induced in Sprague-Dawley rats by withdrawing blood to reduce the mean arterial pressure to 40 mm Hg for 60 minutes. CsA (10 microL/mL) was given 10 minutes before the shock period. The mesenteric postcapillary venules of the proximal ileum were monitored for permeability changes using intravital microscopy. The changes in mitochondrial transmembrane potential were determined using the cationic dye JC-1. Mitochondrial release of cytochrome c in to the cytosol was detected using ELISA. Caspase-3 activity was measured using a fluorometric assay. RESULTS HS induced vascular hyperpermeability, release of cytochrome c, and activation of caspase-3 (p < 0.05). CsA (10 microL/mL) attenuated HS-induced hyperpermeability (p < 0.05) and prevented HS-induced decrease in mitochondrial transmembrane potential. CsA treatment decreased the HS-induced rise in cytosolic cytochrome c levels and caspase-3 activity (p < 0.05). CONCLUSIONS These findings demonstrate that CsA protects mitochondrial permeability transition pores to prevent HS-induced release of cytochrome c and caspase-3 activation.

Mitochondrial complex III is involved in proapoptotic BAK-induced microvascular endothelial cell hyperpermeability.

It has been shown that the intrinsic mitochondrial apoptotic cascade is activated in vascular hyperpermeability after conditions such as hemorrhagic shock. Studies from our laboratory demonstrated mitochondrial reactive oxygen species (ROS) formation in endothelial cells during vascular hyperpermeability. We hypothesized that the participation of mitochondrial ROS in the intrinsic apoptotic cascade results in microvascular endothelial cell hyperpermeability. The purpose of this study was to identify the site(s) of ROS formation in the mitochondrial complex(es) that leads to hyperpermeability. Rat lung microvascular endothelial cell monolayers were pretreated with inhibitors of the complex(es) (I-V) before the activation of the mitochondrial apoptotic cascade using the proapoptotic peptide BAK (BH3). Inhibitors of the xanthine oxidase, nicotinamide adenine dinucleotide phosphate (reduced form) oxidase, NOS, and cytochrome P-450 monooxygenase were also studied. The hyperpermeability was determined by the fluorescence of fluorescein isothiocyanate-albumin that leaked across endothelial cells and ROS production by 2′,7& rime;-dichlorofluorescein diacetate. Cytochrome c levels were also measured. BAK (BH3)-transfected cells showed increased ROS, cytosolic cytochrome c, and hyperpermeability (P < 0.05). Complex III inhibitors antimycin A (10 μM) and stigmatellin (10 μM) attenuated BAK (BH3)-mediated ROS formation and hyperpermeability (P < 0.05). The complex III inhibition decreased BAK (BH3)-mediated cytochrome c release. The results suggest that mitochondrial ROS formation, particularly at respiratory chain complex III, is involved in BAK-induced monolayer hyperpermeability.

β-Catenin dynamics in the regulation of microvascular endothelial cell hyperpermeability.

ABSTRACT &bgr;-Catenin, a key regulator of barrier integrity, is an important component of the adherens junctional complex. Although the roles of &bgr;-catenin in maintaining the adherens junctions and Wnt signaling are known, the dynamics of &bgr;-catenin following insult and its potential role in vascular recovery/repair remain unclear. Our objective was to define &bgr;-catenin’s dynamics following disruption of the adherens junctional complex and subsequent recovery. Rat lung microvascular endothelial cells were treated with active caspase 3 enzyme, by protein transference method, as an inducer of junctional damage and permeability. The disruption and subsequent recovery of &bgr;-catenin to the adherens junctions were studied via immunofluorescence. Rat lung microvascular endothelial cell monolayers were used to measure hyperpermeability. To understand the role of &bgr;-catenin on nuclear translocation/transcriptional regulation in relationship to the recovery of the adherens junctions, Tcf-mediated transcriptional activity was determined. Active caspase 3 induced a loss of &bgr;-catenin at the adherens junctions at 1 and 2 h followed by its recovery at 3 h. Transference of Bak peptide, an inducer of endogenous caspase 3 activation, induced hyperpermeability at 1 h followed by a significant decrease at 2 h. Inhibition of GSK-3&bgr; and the transfection of &bgr;-catenin vector increased Tcf-mediated transcription significantly (P < 0.05). The dissociated adherens junctional protein &bgr;-catenin translocates into the cytoplasm, resulting in microvascular hyperpermeability followed by a time-dependent recovery and relocation to the cell membrane. Our data suggest a recycling pathway for &bgr;-catenin to the cell junction.

Role of β-catenin in regulating microvascular endothelial cell hyperpermeability.

BACKGROUND Paracellular microvascular hyperpermeability occurs mainly because of the disruption of the endothelial adherens junction complex. Vascular endothelial-cadherin that consists of an extracellular and intracellular domain to confer cell-cell contact is linked to the actin cytoskeletal assembly through β-catenin. Our objective was to determine the functional role of β-catenin during paracellular hyperpermeability and to evaluate whether exogenous β-catenin would protect against vascular leak. METHODS β-Catenin siRNA (2.5 μg/mL) was administered to Sprague-Dawley rats through tail vein. FITC-albumin extravasation of the mesenteric postcapillary venules was evaluated after 48 hours using intravital microscopy. Parallel studies using rat lung microvascular endothelial cell monolayers were transfected with β-catenin siRNA, and hyperpermeability was determined using monolayers after 48 hours. The effectiveness of β-catenin siRNA was tested using immunofluorescence and Western blot. To study the protective effect of β-catenin, rat lung microvascular endothelial cell monolayers were transfected with a β-catenin gene expression construct for 48 hours or a recombinant β-catenin protein (1 μg/mL) for 2 hours, followed by transfection with proapoptotic BAK peptide (5 μg/mL), a known inducer hyperpermeability. RESULTS β-Catenin siRNA induced a significant increase in vascular hyperpermeability in vivo (p<0.05) and monolayer permeability (in vitro; p<0.05). β-Catenin siRNA significantly altered the adherens junction complex and decreased β-catenin protein levels. β-Catenin gene expression construct or recombinant β-catenin protein attenuated BAK-induced monolayer hyperpermeability significantly (p<0.05). CONCLUSION Posttranscriptional gene silencing of β-catenin leads to vascular hyperpermeability in vivo and monolayer hyperpermeability in vitro. The enhancement of β-catenin gene expression at the adherens junction or exogenous introduction of β-catenin protein shows protection against vascular hyperpermeability.