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Preconditioning with endoplasmic reticulum stress ameliorates endothelial cell inflammation.

Leonard A, Paton AW, El-Quadi M, Paton JC, Fazal F - PLoS ONE (2014)

Bottom Line: We found that preconditioning human pulmonary artery endothelial cells (HPAEC) to ER stress either by depleting ER chaperone and signaling regulator BiP using siRNA, or specifically cleaving (inactivating) BiP using subtilase cytotoxin (SubAB), alleviates EC inflammation.Mechanistic analysis revealed that pretreatment of EC with SubAB interfered with the binding of the liberated NF-κB to the DNA, thereby resulting in reduced expression of adhesion molecules, cytokines and chemokines.In addition, both knockdown and inactivation of BiP stimulated actin cytoskeletal reorganization resulting in restoration of endothelial permeability.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Lung Biology and Disease Program, University of Rochester School of Medicine and Dentistry, Rochester, New York, United States of America.

ABSTRACT
Endoplasmic Reticulum (ER) stress, caused by disturbance in ER homeostasis, has been implicated in several pathological conditions such as ischemic injury, neurodegenerative disorders, metabolic diseases and more recently in inflammatory conditions. Our present study aims at understanding the role of ER stress in endothelial cell (EC) inflammation, a critical event in the pathogenesis of acute lung injury (ALI). We found that preconditioning human pulmonary artery endothelial cells (HPAEC) to ER stress either by depleting ER chaperone and signaling regulator BiP using siRNA, or specifically cleaving (inactivating) BiP using subtilase cytotoxin (SubAB), alleviates EC inflammation. The two approaches adopted to abrogate BiP function induced ATF4 protein expression and the phosphorylation of eIF2α, both markers of ER stress, which in turn resulted in blunting the activation of NF-κB, and restoring endothelial barrier integrity. Pretreatment of HPAEC with BiP siRNA inhibited thrombin-induced IκBα degradation and its resulting downstream signaling pathway involving NF-κB nuclear translocation, DNA binding, phosphorylation at serine536, transcriptional activation and subsequent expression of adhesion molecules. However, TNFα-mediated NF-κB signaling was unaffected upon BiP knockdown. In an alternative approach, SubAB-mediated inactivation of NF-κB was independent of IκBα degradation. Mechanistic analysis revealed that pretreatment of EC with SubAB interfered with the binding of the liberated NF-κB to the DNA, thereby resulting in reduced expression of adhesion molecules, cytokines and chemokines. In addition, both knockdown and inactivation of BiP stimulated actin cytoskeletal reorganization resulting in restoration of endothelial permeability. Together our studies indicate that BiP plays a central role in EC inflammation and injury via its action on NF-κB activation and regulation of vascular permeability.

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BiP knockdown potentiates actn filment formation and regulates thrombin-induced endothelial permeability.HPAEC were transfected with (A) control-siRNA or (B) BiP-siRNA using DharmaFect1. After 24–36 h cells were fixed, permeabilized, and stained with Alexa Fluor 488 labeled phalloidin to visualize the actin filaments. Images were analyzed by Fluorescence microscopy. Results are representative of three experiments. (C) HPAEC transfected with control-siRNA or BiP- siRNA were seeded at 20,000 cells per transwell insert and cultured for 48 hours. Following this, the confluent monolayer was treated with thrombin (5 U/ml) for 30 minutes. FITC-Dextran permeability testing was done to check monolayer integrity. Permeation was stopped by removing the inserts from the wells. Media from the receiver tray was transferred to a 96 well opaque plate to measure fluorescence. Fluorescent intensities were quantified using a fluorescent plate reader with filters appropriate for 485 nm and 535 nm excitation and emission. The data are the means ± S.E. (n = 3–6 for each condition). #p<0.05 difference from controls; *p<0.05 difference from thrombin-stimulated controls. (D) Following permeability testing the endothelial monolayer transfected with control siRNA (a & b) or with BiP siRNA (c & d), followed by treatment with thrombin (b & d) or left untreated (a & c) were stained for bright field imaging.
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pone-0110949-g008: BiP knockdown potentiates actn filment formation and regulates thrombin-induced endothelial permeability.HPAEC were transfected with (A) control-siRNA or (B) BiP-siRNA using DharmaFect1. After 24–36 h cells were fixed, permeabilized, and stained with Alexa Fluor 488 labeled phalloidin to visualize the actin filaments. Images were analyzed by Fluorescence microscopy. Results are representative of three experiments. (C) HPAEC transfected with control-siRNA or BiP- siRNA were seeded at 20,000 cells per transwell insert and cultured for 48 hours. Following this, the confluent monolayer was treated with thrombin (5 U/ml) for 30 minutes. FITC-Dextran permeability testing was done to check monolayer integrity. Permeation was stopped by removing the inserts from the wells. Media from the receiver tray was transferred to a 96 well opaque plate to measure fluorescence. Fluorescent intensities were quantified using a fluorescent plate reader with filters appropriate for 485 nm and 535 nm excitation and emission. The data are the means ± S.E. (n = 3–6 for each condition). #p<0.05 difference from controls; *p<0.05 difference from thrombin-stimulated controls. (D) Following permeability testing the endothelial monolayer transfected with control siRNA (a & b) or with BiP siRNA (c & d), followed by treatment with thrombin (b & d) or left untreated (a & c) were stained for bright field imaging.

Mentions: Previous work in our lab has shown that the actin cytoskeleton is a dynamic structure that undergoes rearrangement upon exposure to thrombin. Thrombin engages actin depolymerizing protein cofilin, as well as cofilin kinase LIMK1 and cofilin phosphatase SSH-1L to regulate actin dynamics [38], [40] in endothelial cells. The endothelial cell cytoskeleton is primarily composed of three structures: actin microfilaments, microtubules and intermediate filaments. It is the actin filaments that are of critical importance to EC permeability and their role is much more defined as compared to microtubules and intermediate filaments [46]. These studies prompted us to assess the role of BiP in regulating actin cytoskeletal dynamics and endothelial barrier integrity leading to vascular permeability. Controls as well as BiP-depleted cells were stained with Alexa Flour 488-labeled phalloidin to visualize actin filaments. Results show an increase in filamentous actin formation in cells depleted of BiP as compared to control cells (Fig. 8A & B). Furthermore the effect of actin cytoskeleton rearrangement on endothelial permeability was analyzed using an in vitro permeability assay. EC permeability is mostly regulated by intercellular junction organization. The intercellular boundaries in confluent resting cells are maintained by adherence-junction proteins such as VE-cadherin and catenin. Thrombin a serine protease, acts by cleaving its receptor PAR1 causing an increase in intracellular calcium and PKC activation, this in turn stimulates the endothelial contractile apparatus. The endothelial contraction results in disruption of VE-cadherin/catenin complex leading to intercellular gap formation. This effect of thrombin is reversible and is responsible for increased endothelial permeability [47]. To, this end HPAEC transfected with BiP-siRNA or control-siRNA were seeded at 20,000 cells per well into transwell inserts containing 1 µm pores within a transparent polyethylene terephthalate (PET) membrane coated with type 1 rat-tail collagen, and cultured for 48 hours. The confluent monolayer was then treated with thrombin (5 U/ml) for 30 minutes, followed by addition of a high molecular weight FITC-Dextran on top of the cells. The movement of the fluorescent molecule across the endothelial monolayer is a direct determinant of monolayer permeability. Our results show that knockdown of BiP significantly reduced thrombin-induced permeability, as indicated by a marked decrease in the fluorescent counts measured in the receiver tray using a fluorescent plate reader (Fig. 8C). Next the endothelial monolayer was stained to monitor monolayer integrity using Cell stain provided in the kit. Data show that knockdown of BiP significantly reduced the gaps between untreated and thrombin treated cells, implying the role of BiP in EC integrity (Fig. 8D).


Preconditioning with endoplasmic reticulum stress ameliorates endothelial cell inflammation.

Leonard A, Paton AW, El-Quadi M, Paton JC, Fazal F - PLoS ONE (2014)

BiP knockdown potentiates actn filment formation and regulates thrombin-induced endothelial permeability.HPAEC were transfected with (A) control-siRNA or (B) BiP-siRNA using DharmaFect1. After 24–36 h cells were fixed, permeabilized, and stained with Alexa Fluor 488 labeled phalloidin to visualize the actin filaments. Images were analyzed by Fluorescence microscopy. Results are representative of three experiments. (C) HPAEC transfected with control-siRNA or BiP- siRNA were seeded at 20,000 cells per transwell insert and cultured for 48 hours. Following this, the confluent monolayer was treated with thrombin (5 U/ml) for 30 minutes. FITC-Dextran permeability testing was done to check monolayer integrity. Permeation was stopped by removing the inserts from the wells. Media from the receiver tray was transferred to a 96 well opaque plate to measure fluorescence. Fluorescent intensities were quantified using a fluorescent plate reader with filters appropriate for 485 nm and 535 nm excitation and emission. The data are the means ± S.E. (n = 3–6 for each condition). #p<0.05 difference from controls; *p<0.05 difference from thrombin-stimulated controls. (D) Following permeability testing the endothelial monolayer transfected with control siRNA (a & b) or with BiP siRNA (c & d), followed by treatment with thrombin (b & d) or left untreated (a & c) were stained for bright field imaging.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4214695&req=5

pone-0110949-g008: BiP knockdown potentiates actn filment formation and regulates thrombin-induced endothelial permeability.HPAEC were transfected with (A) control-siRNA or (B) BiP-siRNA using DharmaFect1. After 24–36 h cells were fixed, permeabilized, and stained with Alexa Fluor 488 labeled phalloidin to visualize the actin filaments. Images were analyzed by Fluorescence microscopy. Results are representative of three experiments. (C) HPAEC transfected with control-siRNA or BiP- siRNA were seeded at 20,000 cells per transwell insert and cultured for 48 hours. Following this, the confluent monolayer was treated with thrombin (5 U/ml) for 30 minutes. FITC-Dextran permeability testing was done to check monolayer integrity. Permeation was stopped by removing the inserts from the wells. Media from the receiver tray was transferred to a 96 well opaque plate to measure fluorescence. Fluorescent intensities were quantified using a fluorescent plate reader with filters appropriate for 485 nm and 535 nm excitation and emission. The data are the means ± S.E. (n = 3–6 for each condition). #p<0.05 difference from controls; *p<0.05 difference from thrombin-stimulated controls. (D) Following permeability testing the endothelial monolayer transfected with control siRNA (a & b) or with BiP siRNA (c & d), followed by treatment with thrombin (b & d) or left untreated (a & c) were stained for bright field imaging.
Mentions: Previous work in our lab has shown that the actin cytoskeleton is a dynamic structure that undergoes rearrangement upon exposure to thrombin. Thrombin engages actin depolymerizing protein cofilin, as well as cofilin kinase LIMK1 and cofilin phosphatase SSH-1L to regulate actin dynamics [38], [40] in endothelial cells. The endothelial cell cytoskeleton is primarily composed of three structures: actin microfilaments, microtubules and intermediate filaments. It is the actin filaments that are of critical importance to EC permeability and their role is much more defined as compared to microtubules and intermediate filaments [46]. These studies prompted us to assess the role of BiP in regulating actin cytoskeletal dynamics and endothelial barrier integrity leading to vascular permeability. Controls as well as BiP-depleted cells were stained with Alexa Flour 488-labeled phalloidin to visualize actin filaments. Results show an increase in filamentous actin formation in cells depleted of BiP as compared to control cells (Fig. 8A & B). Furthermore the effect of actin cytoskeleton rearrangement on endothelial permeability was analyzed using an in vitro permeability assay. EC permeability is mostly regulated by intercellular junction organization. The intercellular boundaries in confluent resting cells are maintained by adherence-junction proteins such as VE-cadherin and catenin. Thrombin a serine protease, acts by cleaving its receptor PAR1 causing an increase in intracellular calcium and PKC activation, this in turn stimulates the endothelial contractile apparatus. The endothelial contraction results in disruption of VE-cadherin/catenin complex leading to intercellular gap formation. This effect of thrombin is reversible and is responsible for increased endothelial permeability [47]. To, this end HPAEC transfected with BiP-siRNA or control-siRNA were seeded at 20,000 cells per well into transwell inserts containing 1 µm pores within a transparent polyethylene terephthalate (PET) membrane coated with type 1 rat-tail collagen, and cultured for 48 hours. The confluent monolayer was then treated with thrombin (5 U/ml) for 30 minutes, followed by addition of a high molecular weight FITC-Dextran on top of the cells. The movement of the fluorescent molecule across the endothelial monolayer is a direct determinant of monolayer permeability. Our results show that knockdown of BiP significantly reduced thrombin-induced permeability, as indicated by a marked decrease in the fluorescent counts measured in the receiver tray using a fluorescent plate reader (Fig. 8C). Next the endothelial monolayer was stained to monitor monolayer integrity using Cell stain provided in the kit. Data show that knockdown of BiP significantly reduced the gaps between untreated and thrombin treated cells, implying the role of BiP in EC integrity (Fig. 8D).

Bottom Line: We found that preconditioning human pulmonary artery endothelial cells (HPAEC) to ER stress either by depleting ER chaperone and signaling regulator BiP using siRNA, or specifically cleaving (inactivating) BiP using subtilase cytotoxin (SubAB), alleviates EC inflammation.Mechanistic analysis revealed that pretreatment of EC with SubAB interfered with the binding of the liberated NF-κB to the DNA, thereby resulting in reduced expression of adhesion molecules, cytokines and chemokines.In addition, both knockdown and inactivation of BiP stimulated actin cytoskeletal reorganization resulting in restoration of endothelial permeability.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Lung Biology and Disease Program, University of Rochester School of Medicine and Dentistry, Rochester, New York, United States of America.

ABSTRACT
Endoplasmic Reticulum (ER) stress, caused by disturbance in ER homeostasis, has been implicated in several pathological conditions such as ischemic injury, neurodegenerative disorders, metabolic diseases and more recently in inflammatory conditions. Our present study aims at understanding the role of ER stress in endothelial cell (EC) inflammation, a critical event in the pathogenesis of acute lung injury (ALI). We found that preconditioning human pulmonary artery endothelial cells (HPAEC) to ER stress either by depleting ER chaperone and signaling regulator BiP using siRNA, or specifically cleaving (inactivating) BiP using subtilase cytotoxin (SubAB), alleviates EC inflammation. The two approaches adopted to abrogate BiP function induced ATF4 protein expression and the phosphorylation of eIF2α, both markers of ER stress, which in turn resulted in blunting the activation of NF-κB, and restoring endothelial barrier integrity. Pretreatment of HPAEC with BiP siRNA inhibited thrombin-induced IκBα degradation and its resulting downstream signaling pathway involving NF-κB nuclear translocation, DNA binding, phosphorylation at serine536, transcriptional activation and subsequent expression of adhesion molecules. However, TNFα-mediated NF-κB signaling was unaffected upon BiP knockdown. In an alternative approach, SubAB-mediated inactivation of NF-κB was independent of IκBα degradation. Mechanistic analysis revealed that pretreatment of EC with SubAB interfered with the binding of the liberated NF-κB to the DNA, thereby resulting in reduced expression of adhesion molecules, cytokines and chemokines. In addition, both knockdown and inactivation of BiP stimulated actin cytoskeletal reorganization resulting in restoration of endothelial permeability. Together our studies indicate that BiP plays a central role in EC inflammation and injury via its action on NF-κB activation and regulation of vascular permeability.

Show MeSH
Related in: MedlinePlus