Carbachol inhibits TNF-α-induced endothelial barrier dysfunction through alpha 7 nicotinic receptors

Abstract Background: Vascular endothelial barrier function is maintained by cell-to-cell junctional proteins and contributes to vascular homeostasis. Various risk factors such as inflammation disrupt barrier function through down-regulation of these proteins and promote vascular diseases such as atherosclerosis. Previous studies have demonstrated that aged garlic extract (AGE) and its sulfur-containing constituents exert the protective effects against several vascular diseases such as atherosclerosis. In this study, we examined whether AGE and its sulfur-containing constituents improve the endothelial barrier dysfunction elicited by a pro-inflammatory cytokine, Tumor-necrosis factor-α (TNF-α), and explored their mode of action on TNF-α signaling pathway.Methods: Human umbilical vein endothelial cells (HUVECs) were treated with test substances in the presence of TNF-α for various time periods. The endothelial permeability was measured by using a transwell permeability assay. The localization of cell-to-cell junctional proteins and actin cytoskeletons were visualized by immunostaining. RhoA and Rac activities were assessed by using GTP-binding protein pulldown assay. Gene and protein expression levels of signaling molecules were analyzed by real-time PCR and western blotting, respectively. Results: We found that AGE and its major sulfur-containing constituent, S-1-propenylcysteine (S1PC), reduced hyperpermeability elicited by TNF-α in HUVECs. In addition, S1PC inhibited TNF-α-induced production of myosin light chain (MLC) kinase and inactivation of MLC phosphatase through the suppression of the Rac and RhoA signaling pathways, respectively, which resulted in the dephosphorylation of MLC2, a key factor of actin remodeling. Moreover, S1PC inhibited the phosphorylation and activation of guanine nucleotide exchange factor-H1 (GEF-H1), a common upstream key molecule and activator of Rac and RhoA. These effects of S1PC were accompanied by its ability to protect the disruption of junctional proteins on the cell-cell contact regions and the increase of actin stress fibers induced by TNF-α. Conclusions: The present study suggested that AGE and S1PC improve endothelial barrier disruption through the protection of junctional proteins on plasma membrane.

Download Full-text

LBPS 01-15 ASIATIC ACID PROTECTS AGAINST TNF-α-INDUCED ENDOTHELIAL BARRIER DYSFUNCTION IN HUMAN AORTIC ENDOTHELIAL CELLS

Journal of Hypertension ◽

10.1097/01.hjh.0000500385.26032.40 ◽

2016 ◽

Vol 34 (Supplement 1) ◽

pp. e178-e179

Author(s):

Lai Yen Fong ◽

Zuraini Ahmad

Keyword(s):

Endothelial Cells ◽

Endothelial Barrier ◽

Asiatic Acid ◽

Barrier Dysfunction ◽

Aortic Endothelial Cells ◽

Endothelial Barrier Dysfunction ◽

Tnf Α ◽

Human Aortic Endothelial Cells ◽

Aortic Endothelial

Download Full-text

Peroxynitrite mediates TNF-α-induced endothelial barrier dysfunction and nitration of actin

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00391.2005 ◽

2006 ◽

Vol 290 (4) ◽

pp. L674-L684 ◽

Cited By ~ 37

Author(s):

Paul Neumann ◽

Nancy Gertzberg ◽

Erin Vaughan ◽

Joshua Weisbrot ◽

Renee Woodburn ◽

...

Keyword(s):

Tumor Necrosis Factor ◽

Tumor Necrosis ◽

Endothelial Barrier ◽

Barrier Dysfunction ◽

Dot Blot ◽

Endothelial Barrier Dysfunction ◽

Endothelial Monolayers ◽

Tnf Α ◽

Cellular Compartmentalization ◽

Necrosis Factor

We tested the hypothesis that tumor necrosis factor (TNF)-α induces a peroxynitrite (ONOO−)-dependent increase in permeability of pulmonary microvessel endothelial monolayers (PMEM) that is associated with generation of nitrated β-actin (NO2-β-actin). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. β-Actin was extracted from PMEM lysate with a DNase-Sepharose column. The extracted β-actin was quantified in terms of its nitrotyrosine/β-actin ratio with antinitrotyrosine and anti-β-actin antibodies, sequentially, by dot-blot assays. The cellular compartmentalization of NO2-β-actin was displayed by showing confocal localization of nitrotyrosine-immunofluorescence with β-actin-immunofluorescence but not with F-actin fluorescence. Incubation of PMEM with TNF (100 ng/ml) for 0.5 and 4.0 h resulted in increases in permeability to albumin. There was an increase in the nitrotyrosine/β-actin ratio at 0.5 h with minimal association of the NO2-β-actin with F-actin polymers. The TNF-induced increase in the nitrotyrosine/β-actin ratio and permeability were prevented by the anti-ONOO− agent Urate. The data indicate that TNF induces an ONOO−-dependent barrier dysfunction, which is associated with the generation of NO2-β-actin.

Download Full-text

S-1-propenylcysteine improves TNF-α-induced vascular endothelial barrier dysfunction by suppressing the GEF-H1/RhoA/Rac pathway

Cell Communication and Signaling ◽

10.1186/s12964-020-00692-w ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Kayo Kunimura ◽

Satomi Miki ◽

Miyuki Takashima ◽

Jun-ichiro Suzuki

Keyword(s):

Barrier Function ◽

Vascular Diseases ◽

Endothelial Barrier ◽

Major Constituent ◽

Vascular Endothelial ◽

Barrier Dysfunction ◽

Endothelial Barrier Dysfunction ◽

Junctional Proteins ◽

Tnf Α ◽

Sulfur Containing

Abstract Background Vascular endothelial barrier function is maintained by cell-to-cell junctional proteins and contributes to vascular homeostasis. Various risk factors such as inflammation disrupt barrier function through down-regulation of these proteins and promote vascular diseases such as atherosclerosis. Previous studies have demonstrated that aged garlic extract (AGE) and its sulfur-containing constituents exert the protective effects against several vascular diseases such as atherosclerosis. In this study, we examined whether AGE and its sulfur-containing constituents improve the endothelial barrier dysfunction elicited by a pro-inflammatory cytokine, Tumor-necrosis factor-α (TNF-α), and explored their mode of action on TNF-α signaling pathway. Methods Human umbilical vein endothelial cells (HUVECs) were treated with test substances in the presence of TNF-α for various time periods. The endothelial permeability was measured by using a transwell permeability assay. The localization of cell-to-cell junctional proteins and actin cytoskeletons were visualized by immunostaining. RhoA and Rac activities were assessed by using GTP-binding protein pulldown assay. Gene and protein expression levels of signaling molecules were analyzed by real-time PCR and western blotting, respectively. Results We found that AGE and its major sulfur-containing constituent, S-1-propenylcysteine (S1PC), reduced hyperpermeability elicited by TNF-α in HUVECs. In addition, S1PC inhibited TNF-α-induced production of myosin light chain (MLC) kinase and inactivation of MLC phosphatase through the suppression of the Rac and RhoA signaling pathways, respectively, which resulted in the dephosphorylation of MLC2, a key factor of actin remodeling. Moreover, S1PC inhibited the phosphorylation and activation of guanine nucleotide exchange factor-H1 (GEF-H1), a common upstream key molecule and activator of Rac and RhoA. These effects of S1PC were accompanied by its ability to prevent the disruption of junctional proteins on the cell–cell contact regions and the increase of actin stress fibers induced by TNF-α. Conclusions The present study suggested that AGE and its major constituent, S1PC, improve endothelial barrier disruption through the protection of junctional proteins on plasma membrane.

Download Full-text

S-1-propenylcysteine improves TNF-α-induced vascular endothelial barrier dysfunction by suppressing the GEF-H1/RhoA/Rac pathway

10.21203/rs.3.rs-62924/v2 ◽

2020 ◽

Author(s):

Kayo Kunimura ◽

Satomi Miki ◽

Miyuki Takashima ◽

Jun-ichiro Suzuki

Keyword(s):

Barrier Function ◽

Vascular Diseases ◽

Endothelial Barrier ◽

Major Constituent ◽

Vascular Endothelial ◽

Barrier Dysfunction ◽

Endothelial Barrier Dysfunction ◽

Junctional Proteins ◽

Tnf Α ◽

Sulfur Containing

Abstract Background: Vascular endothelial barrier function is maintained by cell-to-cell junctional proteins and contributes to vascular homeostasis. Various risk factors such as inflammation disrupt barrier function through down-regulation of these proteins and promote vascular diseases such as atherosclerosis. Previous studies have demonstrated that aged garlic extract (AGE) and its sulfur-containing constituents exert the protective effects against several vascular diseases such as atherosclerosis. In this study, we examined whether AGE and its sulfur-containing constituents improve the endothelial barrier dysfunction elicited by a pro-inflammatory cytokine, Tumor-necrosis factor-α (TNF-α), and explored their mode of action on TNF-α signaling pathway.Methods: Human umbilical vein endothelial cells (HUVECs) were treated with test substances in the presence of TNF-α for various time periods. The endothelial permeability was measured by using a transwell permeability assay. The localization of cell-to-cell junctional proteins and actin cytoskeletons were visualized by immunostaining. RhoA and Rac activities were assessed by using GTP-binding protein pulldown assay. Gene and protein expression levels of signaling molecules were analyzed by real-time PCR and western blotting, respectively.Results: We found that AGE and its major sulfur-containing constituent, S-1-propenylcysteine (S1PC), reduced hyperpermeability elicited by TNF-α in HUVECs. In addition, S1PC inhibited TNF-α-induced production of myosin light chain (MLC) kinase and inactivation of MLC phosphatase through the suppression of the Rac and RhoA signaling pathways, respectively, which resulted in the dephosphorylation of MLC2, a key factor of actin remodeling. Moreover, S1PC inhibited the phosphorylation and activation of guanine nucleotide exchange factor-H1 (GEF-H1), a common upstream key molecule and activator of Rac and RhoA. These effects of S1PC were accompanied by its ability to prevent the disruption of junctional proteins on the cell-cell contact regions and the increase of actin stress fibers induced by TNF-α.Conclusions: The present study suggested that AGE and its major constituent, S1PC, improve endothelial barrier disruption through the protection of junctional proteins on plasma membrane.

Download Full-text

Selective HDAC6 inhibition prevents TNF-α-induced lung endothelial cell barrier disruption and endotoxin-induced pulmonary edema

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00051.2016 ◽

2016 ◽

Vol 311 (1) ◽

pp. L39-L47 ◽

Cited By ~ 15

Author(s):

Jinyan Yu ◽

Zhongsen Ma ◽

Sreerama Shetty ◽

Mengshi Ma ◽

Jian Fu

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Pulmonary Edema ◽

Fiber Formation ◽

Cell Junctions ◽

Endothelial Barrier ◽

Barrier Dysfunction ◽

Barrier Disruption ◽

Endothelial Barrier Dysfunction ◽

Tnf Α

Lung endothelial damage contributes to the pathogenesis of acute lung injury. New strategies against lung endothelial barrier dysfunction may provide therapeutic benefits against lung vascular injury. Cell-cell junctions and microtubule cytoskeleton are basic components in maintaining endothelial barrier integrity. HDAC6, a deacetylase primarily localized in the cytoplasm, has been reported to modulate nonnuclear protein function through deacetylation. Both α-tubulin and β-catenin are substrates for HDAC6. Here, we examined the effects of tubastatin A, a highly selective HDAC6 inhibitor, on TNF-α induced lung endothelial cell barrier disruption and endotoxin-induced pulmonary edema. Selective HDAC6 inhibition by tubastatin A blocked TNF-α-induced lung endothelial cell hyperpermeability, which was associated with increased α-tubulin acetylation and microtubule stability. Tubastatin A pretreatment inhibited TNF-α-induced endothelial cell contraction and actin stress fiber formation with reduced myosin light chain phosphorylation. Selective HDAC6 inhibition by tubastatin A also induced β-catenin acetylation in human lung endothelial cells, which was associated with increased membrane localization of β-catenin and stabilization of adherens junctions. HDAC6 knockdown by small interfering RNA also prevented TNF-α-induced barrier dysfunction and increased α-tubulin and β-catenin acetylation in endothelial cells. Furthermore, in a mouse model of endotoxemia, tubastatin A was able to prevent endotoxin-induced deacetylation of α-tubulin and β-catenin in lung tissues, which was associated with reduced pulmonary edema. Collectively, our data indicate that selective HDAC6 inhibition by tubastatin A is a potent approach against lung endothelial barrier dysfunction.

Download Full-text