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. Author manuscript; available in PMC: 2014 Sep 1.
Published in final edited form as: J Surg Res. 2013 May 23;184(1):628–637. doi: 10.1016/j.jss.2013.04.079

Regulation of TNF-α-Induced Microvascular Endothelial Cell Hyperpermeability by Recombinant Bcl-xL

Devendra A Sawant 1, Binu Tharakan 2, Rickesha L Wilson 2, Hayden W Stagg 2, Felicia A Hunter 1, Ed W Childs 1,*
PMCID: PMC3759616  NIHMSID: NIHMS477698  PMID: 23731686

Abstract

Background

Tumor necrosis factor-α (TNF-α), a cytotoxic cytokine, induces endothelial cell barrier dysfunction and microvascular hyperpermeability leading to tissue edema, a hall mark of traumatic injuries. The objective of this study was to determine if Bcl-xL, an anti-apoptotic protein, would regulate and protect against TNF-α-mediated endothelial cell barrier dysfunction and microvascular hyperpermeability.

Methods

Rat lung microvascular endothelial cells (RLMECs) were grown as monolayers on Transwell membranes, and FITC-albumin-flux (5mg/ml) across the monolayer was measured fluorometrically to indicate change in monolayer permeability. The RLMEC adherens junctional integrity and actin cytoskeleton were studied by β-catenin immunofluorescence, and using rhodamine phalloidin dye, respectively. Pretreatment of caspase-8 inhibitor (Z-IETD-FMK, 100μM) for 1 hour and transfection of BID siRNA (10μM) for 48 hours, were performed to study their respective effects on TNF-α-induced (10ng/ml; 1 hour treatment) monolayer permeability. Recombinant Bcl-xL protein (2.5μg/ml) was transfected in RLMECs for 1 hour, and its effect on permeability was demonstrated using permeability assay. Caspase-3 activity was assayed fluorometrically.

Results

Z-IETD-FMK pretreatment protected the adherens junctions and decreased TNF-α-induced monolayer hyperpermeability. BID siRNA transfection attenuated 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 demonstrate 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.

Keywords: Apoptotic Signaling, BID, Vascular Permeability, Adherens Junction, β-Catenin

Introduction

Microvascular function is critical for the normal physiology of the circulatory system in the human body.1 Any change in the microvascular permeability could lead to catastrophic events leading to morbidity and mortality in humans.1–5 Microvascular hyperpermeability is commonly observed in many different pathological disorders from inflammatory conditions to ischemia–reperfusion injury, such as hemorrhagic shock, sepsis and thermal injuries.1–5 Furthermore, these conditions are associated with an increase in plasma levels of various cytokines, including tumor necrosis factor-α (TNF-α).1,5,6,7

TNF-α, is a pro-apoptotic/cytotoxic cytokine from TNF superfamily secreted by many different cell types such as macrophages, fibroblasts, smooth muscle cells and endothelial cells.8,9 TNF-α-executes apoptosis through ‘extrinsic’ or receptor mediated apoptotic pathway. TNF-α on binding to its receptor initiates apoptosis by activating caspase cascade involving caspase-8 and caspase-3.9–12 The ‘intrinsic’ or mitochondrial mediated pathway is triggered when the apoptotic signal directly converges on mitochondria. Such stress signals lead to the release of apoptogenic factors, such as cytochrome c from the mitochondrial intermembrane space. The release of cytochrome c into the cytosol leads to effector caspase-3 activation.9,11,12 The ‘extrinsic’ and the ‘intrinsic’ pathways are interlinked through a pro-apoptotic protein, Bcl-2-homology domain 3 (BH3) interacting domain death agonist (BID).9,11–13 The ‘extrinsic’ apoptotic signal brings about activation of caspase-8, which then cleaves cytosolic pro-apoptotic protein BID to truncated BID (tBID). Once tBID is formed, it is translocated from cytosol to mitochondria, and in this process, transfers pro-apoptotic signals from receptors to the mitochondria resulting in the release of pro-apoptotic cytochrome c to initiate apoptosis through downstream activation of effector caspase-3.9,11,12 At the mitochondrial level, the apoptotic signal is neutralized by anti-apoptotic Bcl-2 family of proteins such as B-cell lymphoma-extra large (Bcl-xL).9,11–13

TNF-α is also a major pro-inflammatory cytokine involved in endothelial cell activation, barrier dysfunction and increase in endothelial cell permeability resulting in edema formation.14–16 Endothelial paracellular permeability is regulated by adherens junctional complex proteins such as VE-cadherin and β-catenin.17–20 The adherens junctional protein complex maintain its organization through intracellular attachment to the actin cytoskeleton of the cell.21 Any event leading to disruption of adherens junction protein complex can increase actin stress fiber formation.18,22

In the endothelial cells, TNF-α-induces organizational change in actin cytoskeleton and disruption of adherens junctions leading to an increase in endothelial cell permeability.14–16 Furthermore, results from our laboratory have shown involvement of the ‘intrinsic’ or mitochondrial mediated apoptotic signaling cascade in regulating TNF-α-induced microvascular endothelial cell hyperpermeability. Endothelial cells exposed to TNF-α show an increase in mitochondrial reactive oxygen species (ROS) production as well as a decrease in mitochondrial transmembrane potential, resulting in the release of cytochrome c, caspase-3 activation, and subsequent disruption of adherens junctional protein complex, and increase in endothelial cell permeability (Unpublished data). Active caspase-3 has been shown to cleave or disrupt the adherens junctional complex protein, such as β-catenin, leading to increase in endothelial cell permeability.20,23 Therefore, in this study, we have postulated that Bcl-xL, a mitochondrial anti-apoptotic protein, will attenuate TNF-α-mediated disruption of the adherens junction leading to endothelial cell barrier dysfunction and increase in microvascular permeability.

Materials and Methods

Cell Culture and Reagents

Rat lung microvascular endothelial cells (RLMECs) were obtained from VEC-technologies, Rensselaer, New York. These cells were grown on cell culture dishes coated with 0.1% fibronectin solution from bovine plasma (Sigma-Aldrich, St. Louis, MO) using MCDB-131 complete media obtained from Rensselaer, New York. Trypsin-EDTA solution (0.25%), from Invitrogen-Gibco, Grand Island, NY, was used to detach the RLMECs from fibronectin coated cell culture dishes. Fluorescein isothiocyanate-bovine albumin (FITC-albumin, 5 mg/ml), and recombinant TNF-α (10 ng/ml) were obtained from Sigma-Aldrich, St. Louis, MO. BID siRNA (10 μM), control siRNA along with the siRNA transfection reagent were obtained from Thermo Fisher Scientific – Dharmacon, Lafayette CO. Recombinant Bcl-xL protein (2.5 μg/ml) was obtained from GenScript USA Inc, Piscataway, NJ. Caspase-8 inhibitor, Z-IETD-FMK (100 μM), was obtained from R&D Systems, Minneapolis, MN. Caspase-3 fluoromteric assay kit was obtained from Calbiochem, EMD Millipore, Billerica, MA. BID and β-catenin primary antibodies and anti-rabbit secondary antibodies were obtained from Santa Cruz Biotechnology, Santa Cruz, CA. Rhodamine phalloidin was obtained from Invitrogen, Carlsbad, CA. Vectashield mounting medium containing 4′, 6-diamidino-2-phenylindole (DAPI) was obtained from Vector laboratories, Burlingame, CA. TransIT-LT1 polyamine transfection reagent (10 μl/ml) used for recombinant Bcl-xL protein transfection was obtained from Mirus, Madison, WI.

Effect of caspase-8 inhibitor (Z-IETD-FMK) on TNF-α-induced endothelial cell monolayer hyperpermeability

RLMEC monolayers grown on Transwell plates were used. Sixty minutes prior to the experiments, the monolayers were exposed to fresh media without phenol-red. The following groups were studied: an untreated control group, recombinant TNF-α (10 ng/ml) treated group for 1 hour, recombinant TNF-α (10 ng/ml) treated group for 1 hour along with pre-treatment with Z-IETD-FMK (100 μM) for 1 hour and Z-IETD-FMK alone group treated for 1 hour. The monolayer permeability was determined using FITC-albumin (5 mg/ml) flux as a measure of endothelial cell permeability. During permeability assay, FITC-albumin was added to the upper or luminal chamber of the Transwell plate and was allowed to equilibrate for 30 minutes. The 100 μl of the media samples were collected from the lower or abluminal chambers followed by analysis of fluorescent intensity of FITC using a fluorometric plate reader at excitation/emission 494 nm/520 nm. The data were calculated as percentage of the control values.

Effect of caspase-8 inhibitor (Z-IETD-FMK) on TNF-α-induced disruption of adherens junction

RLMEC monolayers were grown on fibronectin coated chamber slides using MCDB-131 complete media. The following groups were studied: a control or untreated group, recombinant TNF-α (10 ng/ml) treated group, recombinant TNF-α (10 ng/ml) group pre-treated with Z-IETD-FMK (100 μM) and Z-IETD-FMK (100 μM) alone group. All treatments and pretreatments with TNF-α and Z-IETD-FMK, respectively, were carried out for 1 hour each. The cells were fixed in 4% paraformaldehyde and processed for immunofluorescence using a polyclonal antibody against β-catenin overnight at 4°C followed by an FITC-tagged secondary antibody. The cells were mounted using antifade reagent containing DAPI (nuclear stain) and visualized utilizing a confocal laser scanning fluorescent microscope at 60X (Olympus Fluoview).

Effect of BID siRNA transfection on BID immunofluorescence

RLMECs were grown on fibronectin-coated chamber slides. The cells were transfected with BID siRNA (10 μM) using siRNA transfection reagent for 48 hours. After 48 hours, the cells were washed with PBS and fixed in 4% paraformaldehyde. The cells were exposed to 0.5%Triton X-100, washed with PBS and blocked for 1 hour using 2% BSA in PBS. A rabbit polyclonal IgG antibody against BID was applied overnight at 4°C followed by the secondary antibody IgG donkey anti-rabbit FITC. The cells were mounted using DAPI antifade reagent and visualized under a confocal laser scanning fluorescent microscope at 60X (Olympus FluoView).

Effect of BID siRNA on TNF-α-induced endothelial cell monolayer hyperpermeability

To study the role of post-transcriptional gene silencing of BID on TNF-α induced RLMEC monolayer hyperpermeability, we utilized BID specific siRNA. RLMECs were grown as monolayers in fibronectin coated Transwell plates as described above. After 48 hours, the monolayers were transfected with BID siRNA (10 μM) using a siRNA transfection reagent and maintained for 48 hours. After 48 hours, experiments were carried out using the following groups: control or untreated cells, BID siRNA transfected cells, recombinant TNF-α (10 ng/ml) treatment group, cells transfected with BID siRNA exposed to recombinant TNF-α (10 ng/ml) treatment, cells transfected with control siRNA and cells transfected with control siRNA exposed to recombinant TNF-α (10 ng/ml) treatment. The monolayer permeability was determined as described above using FITC-albumin (5 mg/ml) flux as a measure of endothelial cell permeability. The permeability data were calculated as percentage of the control values.

Effect of recombinant Bcl-xL on TNF-α-induced endothelial cell monolayer hyperpermeability

RLMECs were grown as monolayers on fibronectin coated Transwell plates using MCDB-131 complete media for 72 to 96 hours. The following groups were studied: untreated or control group, TNF-α (10 ng/ml) treated group, TransIT-LT1 transfection reagent (10 μl/ml) treated group, TNF-α (10 ng/ml) group pre-treated with Bcl-xL (2.5 μg/ml) transfection and Bcl-xL (2.5 μg/ml) transfected group. All treatments and pretreatments with TNF-α, Bcl-xL and TransIT-LT1, respectively, were carried out for 1 hour each. The monolayer permeability was determined as described above using FITC-albumin (5 mg/ml) flux as a measure of endothelial cell permeability and a fluorometric plate reader was used to read fluorescent intensity at 494/520 nm.

Effect of recombinant Bcl-xL on TNF-α-induced disruption of adherens junctions

RLMECs were grown on fibronectin coated chamber slides using MCDB-131 complete media. The following groups were studied: untreated or control group, TNF-α (10 ng/ml) treated group, TNF-α (10 ng/ml) group pre-treated with Bcl-xL (2.5 μg/ml) and Bcl-xL (2.5 μg/ml) alone group. All treatments and pretreatments with TNF-α and Bcl-xL respectively, were carried out for 1 hour each. The cells were processed for β-catenin immunofluorescence as described above and visualized using a DAPI containing mounting medium under confocal laser scanning fluorescent microscope at 60X (Olympus Fluoview).

Effect of recombinant Bcl-xL on TNF-α-induced disruption of cytoskeletal assembly

RLMECs were grown on fibronectin coated cover glass bottom dishes in complete MCDB-131 media for 24 hours. The following groups were studied: untreated or control group, TNF-α (10 ng/ml) treated group, TNF-α (10 ng/ml) group pre-treated with Bcl-xL (2.5 μg/ml) and Bcl-xL (2.5 μg/ml) alone group. All treatments and pretreatments with TNF-α and Bcl-xL, respectively, were carried out for 1 hour each. The cells were fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100. After blocking with 2.5% BSA-PBS, cells were exposed to rhodamine phalloidin for 30 minutes for visualization of f-actin. The cells were finally mounted in antifade-DAPI reagent and observed under a confocal laser scanning fluorescent microscope at 60X (Olympus Fluoview).

Effect of recombinant Bcl-xL on TNF-α-induced caspase-3 activity

RLMECs were grown on fibronectin coated cell culture dishes in complete MCDB-131 media. The following groups were studied: untreated or control group, TNF-α (10 ng/ml) treated group, TNF-α (10 ng/ml) group pre-treated with Bcl-xL (2.5 μg/ml) and Bcl-xL (2.5 μg/ml) alone group. The RLMECs were lysed by adding sample lysis buffer from the caspase-3 assay kit. After protein estimation, the homogenates were treated with the substrate conjugate labeled with a fluorescent probe. The resulting fluorescent intensity was measured in a fluorescent plate reader using excitation and emission wavelength at 400/505 nm, respectively. Levels of active caspase-3 were assayed using fluorescent intensity values from a fluorescent plate reader.

Statistical analysis

In statistical analysis, all the data are expressed as mean ± SEM. Experimental groups were compared utilizing analysis of variance (ANOVA) followed by the Bonferroni’s post-test for multiple comparisons. A ‘p’ value of ≤ 0.05 was considered to indicate a statistical significant difference. In studies showing monolayer permeability assay, each experimental value was compared with the initial baseline value and expressed as percentage-change.

Results

Caspase-8 inhibitor (Z-IETD-FMK) attenuates TNF-α-induced increase in microvascular endothelial cell hyperpermeability

TNF-α-treated RLMEC monolayers showed an increase in permeability compared with the untreated control monolayers. The FITC albumin fluorescent intensity in the media from abluminal chamber was significantly higher in RLMECs treated with TNF-α (10ng/ml) compared with the control group, suggesting an increase in endothelial cell monolayer permeability (p<0.05). However, the monolayers pre-treated with Z-IETD-FMK followed by TNF-α treatment showed significantly less fluorescent intensity of FITC-albumin in the media from the abluminal chamber (p<0.05; Figure 1A).

Figure 1.

Figure 1

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A) TNF-α-treated monolayers showed significant increase in permeability compared with the untreated control monolayers (*p<0.05). TNF-α-treated monolayers when pre-treated with Z-IETD-FMK showed significant decrease in permeability compared with the TNF-α treated monolayers (a p<0.05). B) TNF-α-treated RLMECs showed discontinuity of β-catenin at cell margins showing the disruption of the adherens junctional complex (indicated by arrows). The RLMECs treated with Z-IETD-FMK prior to the treatment of TNF-α showed protection against adherens junction damage evidenced by the continuity of the β-catenin immunofluorescence at the cell-cell contacts. The blue color indicates DAPI staining for nucleus.

Caspase-8 inhibitor (Z-IETD-FMK) attenuates TNF-α-induced disruption of adherens junctions

TNF-α-treated RLMECs showed intercellular gaps at the cell junction in the form of punctate distribution of β-catenin as compared to continuous distribution of β-catenin at the adherens junctional complex in the control or untreated cells. However, cells pre-treated with Z-IETD-FMK, prevented the TNF-α-induced disruption of the adherens junctional protein complexes in the endothelial cells (Figures 1B).

BID siRNA transfection on BID immunofluorescence

BID siRNA transfected endothelial cells showed a marked decrease in BID immunofluorescence compared to the control or untreated cells and the group with siRNA transfection reagent treated cells (Figure 2A). The decrease in immunofluorescence demonstrates successful siRNA transfection and knockdown of the BID gene from the cells.

Figure 2.

Figure 2

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A) Control/untransfected cells and cells treated with siRNA transfection reagent showed strong BID immunofluorescence. BID siRNA transfected cells showed mark decrease in BID protein evidenced by the decrease in BID immunofluorescence. B) TNF-α-treated monolayers showed significant increase in permeability compared with the untreated control monolayers (*p<0.05). BID siRNA transfected monolayers showed significant decrease in TNF-α-induced increase in monolayer permeability (a p<0.05). Pretreatment with control siRNA transfection failed to show similar effects.

BID siRNA transfection prevents TNF-α-induced endothelial cell monolayer hyperpermeability

TNF-α-treated RLMEC monolayers showed an increase in permeability compared with the untreated control monolayers. The FITC albumin fluorescent intensity in the media from abluminal chamber was significantly higher in RLMECs treated with TNF-α compared with the control group, suggesting an increase in endothelial cell monolayer permeability (p<0.05 Figure 2B). The monolayers transfected with BID siRNA followed by TNF-α treatment showed significantly less fluorescent intensity of FITC-albumin in the media from the abluminal chamber (p<0.05; Figure 2B) compared with TNF-α alone group. However, control siRNA transfected monolayers failed to show similar effect on TNF-α-treated groups. BID siRNA as well as control siRNA transfected monolayers showed no increase in monolayer permeability.

Recombinant Bcl-xL protein attenuates TNF-α-induced endothelial cell monolayer hyperpermeability

TNF-α-treated RLMEC monolayers showed an increase in permeability compared with the untreated control monolayers and recombinant Bcl-xL protein pre-treatment prevented TNF-α-induced increase in endothelial cell permeability. The FITC albumin fluorescent intensity in the media from abluminal chamber was significantly higher in RLMECs treated with TNF-α compared with the control group or TransIT-LT1, transfection reagent treated group suggesting an increase in endothelial cell monolayer permeability (p<0.05; Figure 3A). TransIT-LT1 and Bcl-xL alone treated groups showed no significant increase in monolayer permeability compared with the control group. However, the monolayers pre-treated with recombinant Bcl-xL protein followed by TNF-α treatment showed significantly less fluorescent intensity of FITC-albumin in the media from the abluminal chamber (p<0.05, Figure 3A).

Figure 3.

Figure 3

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A) TNF-α-treated monolayers showed significant increase in permeability compared with the untreated control monolayers (* p<0.05). TNF-α-treated monolayers when pre-treated with Bcl-xL showed significant decrease in permeability compared with the TNF-αtreated monolayers (a p< 0.05). TransIT transfection reagent and Bcl-xL alone treated monolayers showed no increase in permeability. B) TNF-α-treated RLMECs showed discontinuity of β-catenin at cell-cell contact showing the disruption of the barrier (indicated by arrows). The cells treated with Bcl-xL prior to the treatment of TNF-α showed improvement in the junctional damage evidenced by the continuity of the β-catenin immunofluorescence. The blue color indicates DAPI staining for the nucleus.

Recombinant Bcl-xL protects TNF-α-induced disruption of adherens junctions

TNF-α-treated RLMECs showed diffuse punctate distribution of β-catenin in the form of intercellular gaps at the cell junction as compared to continuous distribution of β-catenin at the adherens junctional complex in the control or untreated cells. However, cells pre-treated with Bcl-xL protected the adherens junctional protein complexes from TNF-α-induced disruption, reversing the punctate pattern to characteristics continuous distribution of β-catenin immunofluorescence seen in endothelial cells (Figure 3B).

Recombinant Bcl-xL protects TNF-α-induced disruption of cytoskeletal assembly

TNF-α-treated RLMECs showed increase in actin stress fiber formation compared to the control or untreated cells. However, pre-treatment of cells with Bcl-xL, prevented TNF-α-induced remodeling of actin cytoskeleton and increase in the actin stress fiber formation as evidenced by rhodamine phalloidin staining for f-actin (Figure 4A).

Figure 4.

Figure 4

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A) Rhodamine phalloidin fluorescence showing f-actin cytoskeletal assembly in RLMECs. TNF-α-treated cells showed increase in actin stress fiber formation intracellularly (indicated by arrows). The pre-treatment with Bcl-xL prevented TNF-α-induced actin stress fiber formation. The control cells or untreated cells show no visible increase in stress fiber formation. The blue color indicates DAPI staining for the nucleus. B) TNF-α-induces caspase-3 activity in RLMECs. TNF-α-treated RLMECs show increase in caspase-3 activity compared to untreated control RLMECs (* p<0.05). The Bcl-xL pre-treatment, decreases TNF-α-induced increase in caspase-3 activity significantly in RLMECs (a p<0.05).

Recombinant Bcl-xL decreases TNF-α induced increase in caspase-3 activity

TNF-α-treated RLMECs showed a significant increase in caspase-3 activity when compared with the control or untreated cells (p<0.05; Figure 4B). Bcl-xL pretreatment significantly reduced the TNF-α-induced increase in caspase-3 activity (p<0.05; Figure 4B).

Discussion

Inappropriate inflammatory response during hemorrhagic shock results in to post-injury multiple organ failure, which account for nearly 51–61% of late deaths in trauma patients.5 TNF-α, being a major pro-inflammatory cytokine, is up-regulated in the hemorrhagic shock induced ischemia–reperfusion injury, ensuing microvascular hyperpermeability.4–7 However, the exact pathophysiological pathway behind TNF-α-induced microvascular hyperpermeability is not clearly known. Therefore, the findings from this study have put forward a possible mechanism underlying the TNF-α-induced microvascular hyperpermeability regulated by the ‘extrinsic’ and the ‘intrinsic’ apoptotic signaling pathways.

In this study, we have demonstrated that TNF-α-induces increase in permeability in RLMEC monolayers. The cells pre-treated with caspase-8 inhibitor (Z-IETD-FMK), showed decrease in TNF-α-induced endothelial cell hyperpermeability and reversal of disruption of adherens junction. In RLMECs, BID siRNA transfection showed decrease in BID immunofluorescence indicating post-translational gene silencing of BID protein. We have also demonstrated that BID siRNA transfected RLMECs showed protection against TNF-α-induced endothelial cell hyperpermeability. To protect mitochondrial function from TNF-α-induced apoptotic stimulus, a known mitochondrial specific anti-apoptotic protein Bcl-xL was used. RLMECs transfected with recombinant Bcl-xL protein using TransIT-LT1 polyamine transfection reagent showed protection against TNF-α-induced increase in caspase-3 activity, disruption of adherens junctional protein complex, and increase in actin stress fiber formation. In addition to these safeguards, Bcl-xL also attenuated TNF-α-induced endothelial cell hyperpermeability.

TNF-α on binding to cell surface receptor, triggers intracellular apoptotic signal transduction by creating a DISC complex and causing activation of caspase-8 from procaspase-8 by dimerization.9–12 Active caspase-8 can also initiate ‘intrinsic’ or mitochondrial mediated apoptotic pathway by cleaving BID to form truncated BID (tBID). 9–12 Once tBID is formed in the cytoplasm, it is translocated to the mitochondrial membrane. Insertion of tBID may form pores in the outer membrane leading to mitochondrial outer membrane permeabilization.12,24 In addition, researchers have also shown that tBID is capable of destabilizing lipid membranes in vitro.24,25 Futhermore, tBID also leads to oligomerization of BAX and BAK pro-apoptotic proteins, which forms pores in mitochondrial outer membrane resulting in release of proapoptotic proteins, such as cytochrome c from inner mitochondrial space into the cytosol.12,26–28 The other possibility is that tBID interacts with cardiolipin, a mitochondrial phopholipid, and alters the activity of the respiratory enzymes involved in mitochondrial oxidative phosphorylation resulting in ROS production.12,28,29 This can result in mitochondrial lipid peroxidation, remodeling of mitochondrial cristae, mitochondrial outer membrane permeabilization releasing cytochrome c in to the cytosol, and activating the downstream caspase cascade. 12,24,25,28,29

Mitochondria are protected from the pro-apoptotic stimuli mediated through different pathways by anti-apoptotic proteins, such as Bcl-xL. Bcl-xL is primarily a mitochondrial anti-apoptotic protein. The transmembrane domain of Bcl-xL protein consists of two basic aminoacids at both ends, which allows Bcl-xL to locate specifically on the outer mitochondrial membrane. Therefore, during an apoptotic event, Bcl-xL specifically acts on mitochondria to protect its physiology and maintain its normal function.30,31 Bcl-xL can attenuate apoptotic signaling either by inhibiting the attachment of BAX and BAK to the mitochondrial membrane or by binding directly to BAX and BAK on the mitochondrial membrane, and thereby, preventing their oligomerization to promote cell survival.12,30,32 Furthermore, Bcl-xL, through its interaction with IP3R and/or mitochondrial multiple conductance channels, can reduce Ca2+ spikes, so that there is less movement of Ca2+ in to mitochondria and thus, decreases subsequent mitochondrial events, such as increase in ROS production and attenuate the ‘intrinsic’ or mitochondria mediated apoptotic signaling pathway.30,33

We have summarized the results from the current study in the form of a schematic diagram (Figure 5). In the schematic diagram, we have tried to outline all the important findings of the experiments conducted in the research study through a pathway depicting possible mechanism behind the pathophysiology of TNF-α-induced microvascular endothelial cell hyperpermeability. We have shown that TNF-α-induces microvascular endothelial cell hyperpermeability by activation of caspase-8 through receptor mediated apoptotic signal induction. This was followed by signal transduction through BID translocation from cytosol to the mitochondrial membrane, thus transferring the apoptotic signal from the cell surface receptor to the mitochondria resulting in to activation of effector caspase-3 via release of mitochondrial apoptogenic factors, such as cytochrome c. The active caspase-3 might execute disruption of adherens junctional protein complex and organizational change in the actin cytoskeleton leading to an increase in microvascular endothelial cell permeability. Finally, as a therapeutic approach, we have also demonstrated the protective role of mitochondrial anti-apoptotic protein Bcl-xL against TNF-α-induced increase in caspase-3 activation, endothelial cell barrier dysfunction and increase in paracellular permeability. However, we think that additional in vivo, and translational studies are required to make the results from this study effective in the clinical context, so that in the future, Bcl-xL based therapies could be developed against microvascular hyperpermeability by way of protecting the mitochondria from pro-apoptotic stimuli.

Figure 5.

Figure 5

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The schematic diagram illustrates that TNF-α on binding to its specific cell surface receptor, leads to caspase-8 activation which cleaves cytosolic BID to truncated BID (tBID). Translocation of cytosolic tBID to mitochondrial membrane triggers intrinsic apoptotic signaling resulting in release of cytochrome c, which in turn executes the final step in the apoptotic pathway by activating caspase-3. The active caspase-3 then disrupts the adherens junctional protein complex, along with causing organizational changes in the actin cytoskeletal framework compromising barrier function of the endothelial cells. Mitochondrial anti-apoptotic proteins such as Bcl-xL can protect the mitochondrial physiology from the apoptotic stimulus and thus attenuate the downstream apoptotic cascade.

The significance of this study is that, our findings reaffirm our previous observations on the permissive role of the ‘intrinsic’ apoptotic signaling in hemorrhagic shock induced microvascular hyperpermeability. In a clinical context, microvascular hyperpermeability during hemorrhagic shock, septic shock and burn trauma can lead to more serious consequences such as acute respiratory distress syndrome, abdominal-compartment syndrome, systemic inflammatory response syndrome, and multiple organ dysfunction syndrome.1–5 TNF-α is known to be an important mediator of the pathophysiology of these complications.1,5,6,7 Our findings support a potential link between the ‘intrinsic’ and the ‘extrinsic’ apoptotic signaling pathways mediated via the proapoptotic BID protein and regulated by the cell-surface receptor for TNF-α leading to mitochondrial dysfunction. While this data confirm the role of mitochondria in TNF-α-induced endothelial cell hyperpermeability, it also supports the potential use of mitochondrial specific anti-apoptotic protein Bcl-xL to protect mitochondrial integrity.

Bcl-xL has been associated with many cancerous conditions due to its anti-apoptotic and pro-survival properties.34,35 Extra care should be taken prior to the use of Bcl-xL in the management of microvascular hyperpermeability. However, as microvascular hyperpermeability is an acute process, there is a good possibility of using such anti-apoptotic proteins for short-term treatment or as a pre-treatment during conditions such as hemorrhagic shock to protect mitochondria from the elevated levels of cytokines. As a future direction, this study suggests the use of Bcl-xL molecule with favorable structural alterations, or use of only the active site of Bcl-xL protein, which can be further used for targeted drug delivery, for the acute treatment of microvascular hyperpermeability, without having the long-term deleterious side-effects of Bcl-xL on other organ systems.

Acknowledgments

We acknowledge the Texas A&M Health Science Center College of Medicine Integrated Microscopy and Imaging Laboratory for the use of confocal laser scanning microscope.

Source of Funding

This work was supported by a grant (1K01HL07815-01A1), from National Heart, Lung and Blood Institute, National Institutes of Health, USA.

Footnotes

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