ETS-Related Gene Activation Preserves Adherens Junctions and Permeability in Microvascular Endothelial Cells.

ABSTRACT: ERG (ETS-related gene) is a member of the ETS (Erythroblast-transformation specific) family of transcription factors abundantly present in vascular endothelial cells. Recent studies demonstrate that ERG has important roles in blood vessel stability and angiogenesis. However, it is unclear how ERG is potentially involved in microvascular barrier functions and permeability. A wide variety of diseases and clinical conditions including trauma-hemorrhagic shock and burn injury are associated with microvascular dysfunctions, which causes excessive microvascular permeability, tissue edema and eventually, multiple organ dysfunction and death. The main purpose of this study was to determine the specific role of ERG in regulating microvascular permeability in human lung microvascular endothelial cells (HLMEC) and to evaluate if exogenous ERG will protect the barrier. The HLMECs were grown on Transwell inserts as monolayers and were transfected with ERG CRISPR/cas9 knockdown plasmid, ERG CRISPR activation plasmid, recombinant ERG protein or their respective controls. Recombinant vascular endothelial growth factor (VEGF) was used as an inducer of permeability for evaluating the effect of ERG activation on permeability. Changes in barrier integrity and permeability were studied using monolayer permeability assay and immunofluorescence of adherens junction proteins (VE-cadherin and ß-catenin) respectively. CRISPR/cas9-based ERG knockdown as well as VEGF treatment induced monolayer hyperpermeability, VE-cadherin, and ß-catenin junctional relocation and cytoskeletal F-actin stress fiber formation. CRISPR based ERG activation and recombinant ERG transfection attenuated VEGF-induced monolayer hyperpermeability. ERG activation preserved the adherens junctions and cytoskeleton. These results demonstrate that ERG is a potent regulator of barrier integrity and permeability in human lung microvascular endothelial cells and endogenously or exogenously enhancing ERG provides protection against barrier dysfunction and hyperpermeability.

Protective effects of FK 506 against haemorrhagic shock-induced microvascular hyperpermeability.

Microvascular hyperpermeability, the excessive leakage of fluid and proteins from the intravascular space to the interstitium, is a devastating clinical concern in haemorrhagic shock (HS), sepsis, burn and so forth. Previous studies have shown that HS-induced microvascular hyperpermeability is associated with activation of the mitochondria-mediated 'intrinsic' apoptotic signalling cascade and caspase-3 mediated disruption of the endothelial cell barrier. In this study, our objective was to test if FK506, an immunomodulator that is also known to protect mitochondria, would protect barrier functions and decrease vascular hyperpermeability following HS by acting on this pathway. FK506 (25 µM) was given 10 minutes before the shock period in a rat model of HS. The HS model was a non-traumatic/fixed pressure model of hypovolemic shock developed by withdrawing blood to reduce the mean arterial pressure to 40 mm Hg for 60 minutes. The mesenteric post-capillary venules were monitored for changes in permeability using intravital microscopic imaging. The changes in mitochondrial transmembrane potential (MTP) were determined using the cationic dye 5,5',6,6' tetrachoro-1,1',3,3' tetraethyl benzimidazolyl carbocyanine iodide (JC-1), that was superfused on the mesenteric vasculature followed by intravital imaging. The mesenteric caspase-3 activity was measured fluorometrically. Haemorrhagic shock induced a significant increase in hyperpermeability compared to the sham-control group and FK506 treatment decreased HS-induced hyperpermeability significantly (P < .05). FK506 dampened HS-induced loss of MTP and elevation of caspase-3 activity significantly (P < .05). FK506 has protective effects against HS-induced microvascular hyperpermeability. The maintenance of the MTP and protection against caspase-3 mediated endothelial cell barrier disruption are possible mechanisms by which FK506 attenuates HS-induced hyperpermeability. FK506, currently used in clinical settings as an immunomodulator, needs to be explored further for its therapeutic usefulness against HS-induced vascular hyperpermeability and associated complications.

Glycogen synthase kinase 3 inhibitor protects against microvascular hyperpermeability following hemorrhagic shock.

BACKGROUND: Hemorrhagic shock (HS)-induced microvascular hyperpermeability involves disruption of endothelial cell adherens junctions leading to increase in paracellular permeability. ß-Catenin, an integral component of the adherens junctional complex and Wnt pathway, and caspase 3 via its apoptotic signaling regulate endothelial cell barrier integrity. We have hypothesized that inhibiting phosphorylation of ß-catenin and caspase 3 activity using glycogen synthase kinase 3-specific inhibitor SB216763 would attenuate microvascular hyperpermeability following HS. METHODS: In Sprague-Dawley rats, HS was induced by withdrawing blood to reduce mean arterial pressure to 40 mm Hg for 60 minutes followed by resuscitation. Rats were given SB216763 (600 µg/kg) intravenously 10 minutes before shock. To study microvascular permeability, the rats were intravenously injected with fluorescein isothiocyanate (FITC)-albumin (50 mg/kg), and its flux across the mesenteric postcapillary venules was determined using intravital microscopy. In cell culture studies, rat lung microvascular endothelial cell monolayers grown on Transwell plates were pretreated with SB216763 (5 µM) followed by BAK (5 µg/mL) and caspase 3 (5 µg/mL) protein transfection. FITC-albumin (5 mg/mL) flux across cell monolayers indicates change in monolayer permeability. Activity of canonical Wnt pathway was determined by luciferase assay. Caspase 3 enzyme activity was assayed fluorometrically. RESULTS: The HS group showed significant increase in FITC-albumin extravasation (p < 0.05) compared with sham. SB216763 significantly decrease HS-induced FITC-albumin extravasation (p < 0.05). Pretreatment with SB216763 protected against a BAK-induced increase in rat lung microvascular endothelial cell monolayer permeability and caspase 3 activity but failed to show similar results with a caspase 3-induced increase in monolayer permeability. Wnt3a treatment showed an increase in ß-catenin-dependent T-cell factor-mediated transcription. CONCLUSION: Inhibiting phosphorylation of ß-catenin and caspase 3 activity using glycogen synthase kinase 3-specific inhibitor SB216763 help regulates HS-induced microvascular hyperpermeability.

RECOMBINANT BCL-XL ATTENUATES VASCULAR HYPERPERMEABILITY IN A RAT MODEL OF HEMORRHAGIC SHOCK.

Following hemorrhagic shock (HS), vascular hyperpermeability i.e. the leakage of fluid, nutrients and proteins into the extravascular space occurs primarily due to the disruption of the endothelial cell-cell adherens junctional complex. Studies from our laboratory demonstrate that activation of the mitochondria mediated 'intrinsic' apoptotic signaling cascade has a significant role in modulating HS-induced hyperpermeability. Here we report the novel use of recombinant Bcl-xL, an anti-apoptotic protein, to control HS-induced vascular hyperpermeability. Our results corroborate involvement of vascular hyperpermeability and apoptotic signaling. Hemorrhagic shock (HS) (mean arterial pressure [MAP] was reduced to 40 mmHg for 60 minutes followed by resuscitation to 90 mmHg for 60 minutes) in rats resulted in vascular hyperpermeability as determined by intra-vital microscopy. Treatment of Bcl-xL (2.5ug/ml of rat blood in non-lipid cationic polymer, i.v.) before, during and even after HS attenuated or reversed HS-induced vascular hyperpermeability significantly (p<0.05). Conversely, treatment using Bcl-xL inhibitors, 2-methoxy antimycin (2-MeOAA) and ABT 737, significantly increased vascular hyperpermeability compared to sham (p<0.05). Bcl-xL treatment also decreased the amount of fluid volume required to maintain a MAP of 90 mmHg during resuscitation (p<0.05). HS resulted in increased mitochondrial ROS formation, reduction of ΔΨm, mitochondrial release of cytochrome c and significant activation of caspase-3 (p<0.05). All of these effects were significantly inhibited by Bcl-xL pre-treatment (p<0.05). Our results show that recombinant Bcl-xL is effective against HS-induced vascular hyperpermeability that appears to be mediated through preservation of ΔΨm and subsequent prevention of caspase-3 activation.

Tumor necrosis factor-alpha-induced microvascular endothelial cell hyperpermeability: role of intrinsic apoptotic signaling

Tumor necrosis factor-alfa (TNF-alfa), a pro-apoptotic cytokine, is involved in vascular hyperpermeability, tissue edema, and inflammation. We hypothesized that TNF-alfa induces microvascular hyperpermeability through the mitochondria-mediated intrinsic apoptotic signaling pathway. Rat lung microvascular endothelial cells grown on Transwell inserts, chamber slides, or dishes were treated with recombinant TNF-alfa (10 ng/ml) in the presence or absence of a caspase-3 inhibitor, Z-DEVD-FMK (100 μM). Fluorescein isothiocyanate (FITC)-albumin (5 mg/ml) was used as a marker of monolayer permeability. Mitochondrial reactive oxygen species (ROS) was determined using dihydrorhodamine 123 and mitochondrial transmembrane potential using JC-1. The adherens junction integrity and actin cytoskeletal organization were studied using β-catenin immunofluorescence and rhodamine phalloidin, respectively. Caspase-3 activity was measured fluorometrically. The pretreatment with Z-DEVD-FMK (100 μM) attenuated TNF-alfa-induced (10 ng/ml) disruption of the adherens junctions, actin stress fiber formation, increased caspase-3 activity, and monolayer hyperpermeability (p < 0.05). TNF-alfa (10 ng/ml) treatment resulted in increased mitochondrial ROS formation and decreased mitochondrial transmembrane potential. Intrinsic apoptotic signaling-mediated caspase-3 activation plays an important role in regulating TNF-α-induced endothelial cell hyperpermeability

Tumor necrosis factor-α-induced microvascular endothelial cell hyperpermeability: role of intrinsic apoptotic signaling.

Tumor necrosis factor-α (TNF-α), a pro-apoptotic cytokine, is involved in vascular hyperpermeability, tissue edema, and inflammation. We hypothesized that TNF-α induces microvascular hyperpermeability through the mitochondria-mediated intrinsic apoptotic signaling pathway. Rat lung microvascular endothelial cells grown on Transwell inserts, chamber slides, or dishes were treated with recombinant TNF-α (10 ng/ml) in the presence or absence of a caspase-3 inhibitor, Z-DEVD-FMK (100 µM). Fluorescein isothiocyanate (FITC)-albumin (5 mg/ml) was used as a marker of monolayer permeability. Mitochondrial reactive oxygen species (ROS) was determined using dihydrorhodamine 123 and mitochondrial transmembrane potential using JC-1. The adherens junction integrity and actin cytoskeletal organization were studied using ß-catenin immunofluorescence and rhodamine phalloidin, respectively. Caspase-3 activity was measured fluorometrically. The pretreatment with Z-DEVD-FMK (100 µM) attenuated TNF-α-induced (10 ng/ml) disruption of the adherens junctions, actin stress fiber formation, increased caspase-3 activity, and monolayer hyperpermeability (p < 0.05). TNF-α (10 ng/ml) treatment resulted in increased mitochondrial ROS formation and decreased mitochondrial transmembrane potential. Intrinsic apoptotic signaling-mediated caspase-3 activation plays an important role in regulating TNF-α-induced endothelial cell hyperpermeability.

The role of intrinsic apoptotic signaling in hemorrhagic shock-induced microvascular endothelial cell barrier dysfunction.

Hemorrhagic shock leads to endothelial cell barrier dysfunction resulting in microvascular hyperpermeability. Hemorrhagic shock-induced microvascular hyperpermeability is associated with worse clinical outcomes in patients with traumatic injuries. The results from our laboratory have illustrated a possible pathophysiological mechanism showing involvement of mitochondria-mediated "intrinsic" apoptotic signaling in regulating hemorrhagic shock-induced microvascular hyperpermeability. Hemorrhagic shock results in overexpression of Bcl-2 family of pro-apoptotic protein, BAK, in the microvascular endothelial cells. The increase in BAK initiates "intrinsic" apoptotic signaling cascade with the release of mitochondrial cytochrome c in the cytoplasm and activation of downstream effector caspase-3, leading to loss of endothelial cell barrier integrity. Thus, this review article offers a brief overview of important findings from our past and present research work along with new leads for future research. The summary of our research work will provide information leading to different avenues in developing novel strategies against microvascular hyperpermeability following hemorrhagic shock.

Doxycycline attenuates burn-induced microvascular hyperpermeability.

BACKGROUND: Burns induce systemic microvascular hyperpermeability resulting in shock, and if untreated, cardiovascular collapse. Damage to the endothelial cell adherens junctional complex plays an integral role in the pathophysiology of microvascular hyperpermeability. We hypothesized that doxycycline, a known inhibitor of matrix metalloproteinases (MMPs), could attenuate burn-induced adherens junction damage and microvascular hyperpermeability. METHODS: Male Sprague-Dawley rats were divided into sham, burn, and burn + doxycycline (n = 5). The experimental groups underwent a 30% total body surface area full-thickness burn. Fluorescein isothiocyanate-albumin was administered intravenously. Mesenteric postcapillary venules were examined with intravital microscopy to determine flux of albumin from the intravascular space to the interstitium. Fluorescence intensity was compared between the intravascular space to the interstitium at 30, 60, 80, 100, 120, 140, 160, and 180 minutes after burn. Parallel experiments were performed in which rat lung microvascular endothelial cells were treated with sera from sham or burn animals as well as separate groups pretreated with either doxycycline or a specific inhibitor of MMP-9. Monolayer permeability was determined by fluorescein isothiocyanate albumin-flux across Transwell plates and immunofluorescense staining for the adherens junction protein ß-catenin was performed. Western blot and gelatin zymography were performed to assess MMP-9 level and activity. RESULTS: MMP-9 levels were increased after burn. Monolayer permeability was significantly increased with burn serum treatment; this was attenuated with doxycycline as well as the specific MMP-9 inhibitor (p < 0.05). Damage of the endothelial cell adherens junction complex was induced by serum from burned rats, and doxycycline restored the integrity of the adherens junction similar to the MMP-9 inhibitor. Intravital microscopy revealed microvascular hyperpermeability after burn; this was attenuated with doxycycline (p < 0.05). CONCLUSION: Burns induce microvascular hyperpermeability via endothelial adherens junction disruption associated with MMP-9, and this is attenuated with doxycycline.

Regulation of tumor necrosis factor-α-induced microvascular endothelial cell hyperpermeability by recombinant B-cell lymphoma-extra large.

BACKGROUND: Tumor necrosis factor-α (TNF-α), a cytotoxic cytokine, induces endothelial cell barrier dysfunction and microvascular hyperpermeability, leading to tissue edema, a hallmark of traumatic injuries. The objective of the present study was to determine whether B-cell lymphoma-extra large (Bcl-xL), an antiapoptotic protein, would regulate and protect against TNF-α-mediated endothelial cell barrier dysfunction and microvascular hyperpermeability. METHODS: Rat lung microvascular endothelial cells were grown as monolayers on Transwell membranes, and fluorescein isothiocyanate-bovine albumin flux (5 mg/mL) across the monolayer was measured fluorometrically to indicate changes in monolayer permeability. The rat lung microvascular endothelial cell adherens junctional integrity and actin cytoskeleton was studied using ß-catenin immunofluorescence and rhodamine phalloidin dye, respectively. Pretreatment of caspase-8 inhibitor (Z-IETD-FMK, 100 µM) for 1 hour and transfection of Bcl-2-homology domain 3-interacting domain death agonist small interfering RNA (10 µM) for 48 hours were performed to study their respective effects on TNF-α-induced (10 ng/mL; 1-hour treatment) monolayer permeability. Recombinant Bcl-xL protein (2.5 µg/ml) was transfected in rat lung microvascular endothelial cells for 1 hour, and its effect on permeability was demonstrated using a permeability assay. Caspase-3 activity was assayed fluorometrically. RESULTS: Z-IETD-FMK pretreatment protected the adherens junctions and decreased TNF-α-induced monolayer hyperpermeability. Bcl-2-homology domain 3-interacting domain death agonist small interfering RNA transfection attenuated the TNF-α-induced increase in monolayer permeability. Recombinant Bcl-xL protein showed protection against TNF-α-induced actin stress fiber formation, an increase in caspase-3 activity, and monolayer hyperpermeability. CONCLUSIONS: Our results have demonstrated the protective effects of recombinant Bcl-xL protein against TNF-α-induced endothelial cell adherens junction damage and microvascular endothelial cell hyperpermeability. These findings support the potential for Bcl-xL-based drug development against microvascular hyperpermeability and tissue edema.

Microvascular endothelial cell hyperpermeability induced by endogenous caspase 3 activator staurosporine.

BACKGROUND: Microvascular hyperpermeability following conditions such as hemorrhagic shock occurs mainly owing to disruption of the adherens junctional protein complex in endothelial cells. The objective of this study was to examine the action of staurosporine, a potent activator of endogenous caspase 3 on the adherens junction and the cellular pathway through which it causes possible endothelial cell barrier dysfunction. METHODS: Rat lung microvascular endothelial cell (RLMEC) permeability was measured by fluorescein isothiocyanate-albumin flux across the monolayer in a Transwell plate. Integrity of the endothelial cell adherens junctions was studied using immunofluorescence of ß-catenin and vascular endothelial-cadherin. Mitochondrial reactive oxygen species formation was determined by using dihydrorhodamine 123 and mitochondrial transmembrane potential by JC-1 fluorescent probe and flow cytometry. Caspase 3 enzyme activity was assayed fluorometrically. Cell death assay in RLMECs was performed using propidium iodide staining and analyzed by flow cytometry. RESULTS: Staurosporine (1 µM)-treated RLMEC monolayers showed significant increase in permeability, which was decreased by pretreatment with caspase 3 specific inhibitor, Z-DEVD-FMK (p < 0.05). Immunofluorescence studies showed staurosporine induced disruption of the adherens junction, which was reversed by Z-DEVD-FMK. Staurosporine treatment led to an increase in mitochondrial reactive oxygen species formation and a decrease in mitochondrial transmembrane potential. Furthermore, staurosporine induced a significant increase in caspase 3 activity (p < 0.05) but not cell death in RLMECs (p < 0.05). CONCLUSION: Staurosporine-induced disruption of the adherens junction and microvascular endothelial cell hyperpermeability is associated with the activation of mitochondrial "intrinsic" apoptotic signaling cascade but without causing endothelial cell death. Our results suggest that prevention of mitochondrial-mediated activation of caspase 3 has therapeutic potential against microvascular hyperpermeability.

Inhibition of Fas-Fas ligand interaction attenuates microvascular hyperpermeability following hemorrhagic shock.

Hemorrhagic shock (HS)-induced microvascular hyperpermeability poses a serious challenge in the management of trauma patients. Microvascular hyperpermeability occurs mainly because of the disruption of endothelial cell adherens junctions, where the "intrinsic" apoptotic signaling plays a regulatory role. The purpose of this study was to understand the role of the "extrinsic" apoptotic signaling molecules, particularly Fas-Fas ligand interaction in microvascular endothelial barrier integrity. Rat lung microvascular endothelial cells (RLMECs) were exposed to HS serum in the presence or absence of the Fas ligand inhibitor, FasFc. The effect of HS serum on Fas receptor and Fas ligand expression on RLMECs was determined by flow cytometry. Endothelial cell permeability was determined by monolayer permeability assay and the barrier integrity by ß-catenin immunofluorescence. Mitochondrial reactive oxygen species formation was determined using dihydrorhodamine 123 probe by fluorescent microscopy. Mitochondrial transmembrane potential was studied by fluorescent microscopy as well as flow cytometry. Caspase 3 enzyme activity was assayed fluorometrically. Rat lung microvascular endothelial cells exposed to HS serum showed increase in Fas receptor and Fas ligand expression levels. FasFc treatment showed protection against HS serum-induced disruption of the adherens junctions and monolayer hyperpermeability (P < 0.05) in the endothelial cells. Pretreatment with FasFc also decreased HS serum-induced increase in mitochondrial reactive oxygen species formation, restored HS serum-induced drop in mitochondrial transmembrane potential, and reduced HS serum-induced caspase 3 activity in RLMECs. These findings open new avenues for drug development to manage HS-induced microvascular hyperpermeability by targeting the Fas-Fas ligand-mediated pathway.

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

ß-Catenin, a key regulator of barrier integrity, is an important component of the adherens junctional complex. Although the roles of ß-catenin in maintaining the adherens junctions and Wnt signaling are known, the dynamics of ß-catenin following insult and its potential role in vascular recovery/repair remain unclear. Our objective was to define ß-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 ß-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 ß-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 ß-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-3ß and the transfection of ß-catenin vector increased Tcf-mediated transcription significantly (P < 0.05). The dissociated adherens junctional protein ß-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 ß-catenin to the cell junction.

Role of ß-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 ß-catenin. Our objective was to determine the functional role of ß-catenin during paracellular hyperpermeability and to evaluate whether exogenous ß-catenin would protect against vascular leak. METHODS: ß-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 ß-catenin siRNA, and hyperpermeability was determined using monolayers after 48 hours. The effectiveness of ß-catenin siRNA was tested using immunofluorescence and Western blot. To study the protective effect of ß-catenin, rat lung microvascular endothelial cell monolayers were transfected with a ß-catenin gene expression construct for 48 hours or a recombinant ß-catenin protein (1 µg/mL) for 2 hours, followed by transfection with proapoptotic BAK peptide (5 µg/mL), a known inducer hyperpermeability. RESULTS: ß-Catenin siRNA induced a significant increase in vascular hyperpermeability in vivo (p<0.05) and monolayer permeability (in vitro; p<0.05). ß-Catenin siRNA significantly altered the adherens junction complex and decreased ß-catenin protein levels. ß-Catenin gene expression construct or recombinant ß-catenin protein attenuated BAK-induced monolayer hyperpermeability significantly (p<0.05). CONCLUSION: Posttranscriptional gene silencing of ß-catenin leads to vascular hyperpermeability in vivo and monolayer hyperpermeability in vitro. The enhancement of ß-catenin gene expression at the adherens junction or exogenous introduction of ß-catenin protein shows protection against vascular hyperpermeability.

Inhibition of VE-cadherin proteasomal degradation attenuates microvascular hyperpermeability.

OBJECTIVE: VE-cadherin, an integral component of the adherens junction complex, is processed through the endosome-lysosome pathway and proteasome system for degradation. Our objective was to determine if inhibition of this pathway would protect against microvascular hyperpermeability. METHODS: To induce VE-cadherin degradation, we utilized a mutant VE-cadherin protein that lacks the extracellular domain (rVE-cad CPD). Intravital microscopy was employed to study the changes in microvascular permeability in rat mesenteric postcapillary venules. Rat lung microvascular endothelial cell (RLMEC) monolayers were utilized in parallel studies. The adherens junction integrity was determined using VE-cadherin and ß-catenin immunofluorescence. TOPflash/FOPflash transfection and luciferase reporter assay were performed to study ß-catenin-mediated transcriptional activation. RESULTS: rVE-cad CPD (2.5 µg/mL of blood volume) increased hyperpermeability significantly (p < 0.05). The VE-cadherin siRNA as well as rVE-cad CPD induced significant increase in monolayer hyperpermeability (p < 0.05). Transfection of rVE-cad CPD disrupted adherens junctions evidenced by discontinuity in ß-catenin and VE-cadherin immunofluorescence (p < 0.05). Proteasome inhibitor MG132 attenuated rVE-cad CPD induced monolayer hyperpermeability and adherens junction damage. CONCLUSIONS: VE-cadherin disruption in animals results in hyperpermeability. Parallel studies in RLMEC demonstrated similar results. In addition, inhibition of proteasomal degradation attenuated microvascular hyperpermeability. These findings have significance in understanding the role of VE-cadherin in regulating vascular hyperpermeability.

Curcumin inhibits reactive oxygen species formation and vascular hyperpermeability following haemorrhagic shock.

1. Oxidative stress induced by reactive oxygen species (ROS) is a key mediator of haemorrhagic shock (HS)-induced vascular hyperpermeability. In the present study, curcumin, a natural anti-oxidant obtained from turmeric (Curcuma longa), was tested against HS-induced hyperpermeability and associated ROS formation in rat mesenteric post-capillary venules in vivo and in rat lung microvascular endothelial cells (RLMEC) in vitro. 2. In rats, HS was induced by withdrawing blood to reduce mean arterial pressure to 40 mmHg for 60 min, followed by resuscitation for 60 min. To investigate vascular permeability, rats were given fluorescein isothiocyanate (FITC)-albumin (50 mg/kg, i.v.). The FITC-albumin flux was measured in mesenteric post-capillary venules by determining optical intensity intra- and extravascularly under intravital microscopy. Mitochondrial ROS formation was determined using dihydrorhodamine 123 in vivo. Parallel studies were conducted in vitro using serum collected after HS. The serum was tested on rat lung microvascular endothelial cell RLMEC monolayers. 3. In rats, HS induced a significant increase in vascular hyperpermeability and ROS formation in vivo (P < 0.05). Treatment with curcumin (20 micromol/L) attenuated both these effects (P < 0.05). In RLMEC in vitro, HS serum induced monolayer permeability and ROS formation. Curcumin (10 micromol/L) attenuated HS serum-induced monolayer hyperpermeability and ROS formation. Curcumin (2-100 micromol/L) scavenged 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and 1,1-diphenyl-2-picrylhydrazyl radicals in vitro, indicating its potential as a free radical scavenger. 4. The present study demonstrates that curcumin is an inhibitor of vascular hyperpermeability following HS, with its protective effects mediated through its anti-oxidant properties.

Cyclosporine A--protection against microvascular hyperpermeability is calcineurin independent.

BACKGROUND: Mitochondria-mediated apoptotic signaling contributes to microvascular hyperpermeability. We hypothesized that cyclosporine A (CsA), which protects mitochondrial transition pores, would attenuate hyperpermeability independent of its calcineurin inhibitory property. METHODS: Hyperpermeability was induced in microvascular endothelial cell monolayers using proapoptotic BAK or active caspase-3 after CsA or a specific calcineurin inhibitor, calcineurin autoinhibitory peptide (CIP), treatment. Permeability was measured based on fluorescein isothiocyanate-albumin flux across the monolayers. Mitochondrial transmembrane potential (MTP) was determined using 5,5',6,6'-tetrachoro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide. Mitochondrial release of cytochrome c was measured using an enzyme-linked immunosorbent assay and caspase-3 activity fluorometrically. RESULTS: CsA-attenuated (10 nmol/L) but not CIP-attenuated (100 mumol/L) BAK induced hyperpermeability (P < .05), CsA- but not CIP-attenuated BAK induced a decrease in MTP and an increase in cytochrome c levels and caspase-3 activity (P < .05). CsA and CIP were ineffective against caspase-3-induced hyperpermeability. CONCLUSIONS: CsA attenuated hyperpermeability by protecting MTP, thus preventing mitochondria-mediated apoptotic signaling. The protective effect of CsA is independent of calcineurin inhibition.

17beta-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 17beta-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 17beta-estradiol, tamoxifen, fulvestrant plus 17beta-estradiol, propyl pyrazole triol plus 17beta-estradiol, and diarylpropionitrile plus 17beta-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',6,6'tetrachloro-1,1',3,3'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 17beta-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 alpha and beta estrogen receptor agonists inhibited HS-induced hyperpermeability (P < 0.05). 17beta-Estradiol decreased HS-induced reactive oxygen species formation and restored mitochondrial transmembrane potential. 17beta-Estradiol decreased both cytosolic cytochrome c level and activation of caspase-3 (P < 0.05). These findings suggest that 17beta-estradiol protects the microvasculature after HS, and that this protection may be mediated through the alpha and beta estrogen receptors.

(-)-Deprenyl inhibits vascular hyperpermeability after hemorrhagic shock.

Recent studies from our laboratory demonstrated the involvement of endothelial cell reactive oxygen species (ROS) formation and activation of apoptotic signaling in vascular hyperpermeability after hemorrhagic shock (HS). The objective of this study was to determine if (-)-deprenyl, an antioxidant with antiapoptotic properties, would attenuate HS-induced vascular hyperpermeability. In rats, HS was induced by withdrawing blood to reduce the MAP to 40 mmHg for 60 min followed by resuscitation for 60 min. To study hyperpermeability, we injected the rats with fluorescein isothiocyanate--albumin (50 mg/kg), and the changes in integrated optical intensity of the mesenteric postcapillary venules were obtained intravascularly and extravascularly using intravital microscopy. Mitochondrial ROS formation and mitochondrial transmembrane potential (DeltaPsim) were studied using dihydrorhodamine 123 and JC-1, respectively. Mitochondrial release of cytochrome c was determined using enzyme-linked immunosorbent assay and caspase-3 activity by a fluorometric assay. Parallel studies were performed in rat lung microvascular endothelial cells using proapoptotic BAK as inducer of hyperpermeability. Hemorrhagic shock induced vascular hyperpermeability, mitochondrial ROS formation, DeltaPsim decrease, cytochrome c release, and caspase-3 activation (P G 0.05). (-)-Deprenyl (0.15 mg/kg) attenuated all these effects (P < 0.05). Similarly in rat lung microvascular endothelial cells, (-)-deprenyl attenuated BAK peptide-induced monolayer hyperpermeability (P < 0.05), ROS formation, DeltaPsim decrease, cytochrome c release (P<0.05), and caspase-3 activation (P < 0.05). The protective effects of (-)-deprenyl on vascular barrier functions may be due to its protective effects on DeltaPsim, thereby preventing mitochondrial release of cytochrome c and caspase-3--mediated disruption of endothelial adherens junctions.

(-)-Deprenyl inhibits thermal injury-induced apoptotic signaling and hyperpermeability in microvascular endothelial cells.

Burn injury is associated with a significant leak of intravascular fluid into the interstitial space, requiring large amounts of volume resuscitation. Activation of the intrinsic (mitochondrial) apoptotic pathway has been associated with vascular hyperpermeability. We hypothesized that vascular hyperpermeability following burns is also mediated via this pathway. The purpose of this study was to investigate whether (-)-deprenyl, a drug with antioxidant and antiapoptotic properties, could attenuate burn induced-apoptotic signaling and hyperpermeability. Male Sprague-Dawley rats were assigned to sham or experimental groups. The experimental rats underwent a 30 to 40% TBSA full-thickness burn. Serum was collected from all rats at 180 minutes postburn. Rat lung microvascular endothelial cell monolayers were exposed to the sham or burn serum; permeability was determined by fluorescein isothiocyanate-tagged albumin flux. Mitochondrial reactive oxygen species formation was measured with dihydrorhodamine 123. The change in mitochondrial membrane potential was determined with JC-1. Cytosolic cytochrome c was measured by enzyme-linked immunosorbent assay. A group of cells in each series was pretreated with (-)-deprenyl (1 microM). Monolayer permeability increased significantly (P<.05) when treated with burn serum. (-)-Deprenyl significantly attenuated the hyperpermeability induced by burn serum (P<.05). Burn serum increased mitochondrial reactive oxygen species levels and reduced mitochondrial membrane potential; these effects were markedly reduced by (-)-deprenyl. Cytochrome c release was increased by treatment with burn serum (P<.05), and this effect was significantly inhibited by (-)-deprenyl (P<.05). Burn serum induces hyperpemeability and activates intrinsic apoptotic signaling in microvascular endothelial cells. (-)-Deprenyl, an antioxidant and antiapoptotic drug, modulates intrinsic apoptotic signaling and attenuates burn-induced hyperpermeability.

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.