Inhibition of interleukin-6 trans-signaling prevents inflammation and endothelial barrier disruption in retinal endothelial cells

Thrombin, a procoagulant protease, cleaves and activates protease-activated receptor-1 (PAR1) to promote inflammatory responses and endothelial dysfunction. In contrast, activated protein C (APC), an anticoagulant protease, activates PAR1 through a distinct cleavage site and promotes anti-inflammatory responses, prosurvival, and endothelial barrier stabilization. The distinct tethered ligands formed through cleavage of PAR1 by thrombin versus APC result in unique active receptor conformations that bias PAR1 signaling. Despite progress in understanding PAR1 biased signaling, the proteins and pathways utilized by thrombin versus APC signaling to induce opposing cellular functions are largely unknown. Here, we report the global phosphoproteome induced by thrombin and APC signaling in endothelial cells with the quantification of 11,266 unique phosphopeptides using multiplexed quantitative mass spectrometry. Our results reveal unique dynamic phosphoproteome profiles of thrombin and APC signaling, an enrichment of associated biological functions, including key modulators of endothelial barrier function, regulators of gene transcription, and specific kinases predicted to mediate PAR1 biased signaling. Using small interfering RNA to deplete a subset of phosphorylated proteins not previously linked to thrombin or APC signaling, a function for afadin and adducin-1 actin binding proteins in thrombin-induced endothelial barrier disruption is unveiled. Afadin depletion resulted in enhanced thrombin-promoted barrier permeability, whereas adducin-1 depletion completely ablated thrombin-induced barrier disruption without compromising p38 signaling. However, loss of adducin-1 blocked APC-induced Akt signaling. These studies define distinct thrombin and APC dynamic signaling profiles and a rich array of proteins and biological pathways that engender PAR1 biased signaling in endothelial cells.

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Interleukin-6 Trans-Signaling Mediated Regulation of Paracellular Permeability in Human Retinal Endothelial Cells

International Journal of Translational Medicine ◽

10.3390/ijtm1020010 ◽

2021 ◽

Vol 1 (2) ◽

pp. 137-153

Author(s):

Joshua Glass ◽

Rebekah Robinson ◽

Tae-Jin Lee ◽

Ashok Sharma ◽

Shruti Sharma

Keyword(s):

Gene Expression ◽

Endothelial Cells ◽

Diabetic Retinopathy ◽

Interleukin 6 ◽

Vascular Inflammation ◽

Paracellular Permeability ◽

Blood Retinal Barrier ◽

Retinal Endothelial Cells ◽

Expression Of Genes ◽

Signaling Activation

Long-term hyperglycemia-mediated oxidative stress and inflammation lead to the blood-retinal barrier (BRB) dysfunction and increased vascular permeability associated with diabetic retinopathy (DR). Interleukin-6 (IL-6) is one of the primary mediators of retinal vascular inflammation. IL-6 signaling through its membrane-bound IL-6 receptor is known as classical signaling, and through a soluble IL-6 receptor (sIL-6R) is known as trans-signaling. Increasing evidence suggests that classical signaling is primarily anti-inflammatory, whereas trans-signaling induces the pro-inflammatory effects of IL-6. The purpose of this study was to compare the effects of these two pathways on paracellular permeability and expression of genes involved in inter-endothelial junctions in human retinal endothelial cells (HRECs). IL-6 trans-signaling activation caused significant disruption to paracellular integrity, with increased paracellular permeability, and was associated with significant changes in gene expression related to adherens, tight, and gap junctions. IL-6 classical signaling did not alter paracellular resistance in HRECs and had no distinct effects on gene expression. In conclusion, IL-6 trans-signaling, but not classical signaling, is a major mediator of the increased paracellular permeability characteristic of inner BRB breakdown in diabetic retinopathy. This study also identified potential inter-endothelial junction genes involved in the IL-6 trans-signaling mediated regulation of paracellular permeability in HRECs.

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Interleukin 6-Mediated Endothelial Barrier Disturbances Can Be Attenuated by Blockade of the IL6 Receptor Expressed in Brain Microvascular Endothelial Cells

Translational Stroke Research ◽

10.1007/s12975-018-0614-2 ◽

2018 ◽

Vol 9 (6) ◽

pp. 631-642 ◽

Cited By ~ 33

Author(s):

Kinga G. Blecharz-Lang ◽

Josephin Wagner ◽

Alexa Fries ◽

Melina Nieminen-Kelhä ◽

Jörg Rösner ◽

...

Keyword(s):

Endothelial Cells ◽

Interleukin 6 ◽

Endothelial Barrier ◽

Microvascular Endothelial Cells ◽

Brain Microvascular Endothelial Cells ◽

Microvascular Endothelial

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Depletion of Arg/Abl2 improves endothelial cell adhesion and prevents vascular leak during inflammation

Angiogenesis ◽

10.1007/s10456-021-09781-x ◽

2021 ◽

Author(s):

Joana Amado-Azevedo ◽

Anne-Marieke D. van Stalborch ◽

Erik T. Valent ◽

Kalim Nawaz ◽

Jan van Bezu ◽

...

Keyword(s):

Endothelial Cells ◽

Rho Gtpase ◽

Endothelial Barrier ◽

Cell Periphery ◽

Gap Formation ◽

Vascular Leak ◽

Barrier Disruption ◽

Cell Matrix ◽

Cell Retraction

AbstractEndothelial barrier disruption and vascular leak importantly contribute to organ dysfunction and mortality during inflammatory conditions like sepsis and acute respiratory distress syndrome. We identified the kinase Arg/Abl2 as a mediator of endothelial barrier disruption, but the role of Arg in endothelial monolayer regulation and its relevance in vivo remain poorly understood. Here we show that depletion of Arg in endothelial cells results in the activation of both RhoA and Rac1, increased cell spreading and elongation, redistribution of integrin-dependent cell-matrix adhesions to the cell periphery, and improved adhesion to the extracellular matrix. We further show that Arg is activated in the endothelium during inflammation, both in murine lungs exposed to barrier-disruptive agents, and in pulmonary microvessels of septic patients. Importantly, Arg-depleted endothelial cells were less sensitive to barrier-disruptive agents. Despite the formation of F-actin stress fibers and myosin light chain phosphorylation, Arg depletion diminished adherens junction disruption and intercellular gap formation, by reducing the disassembly of cell-matrix adhesions and cell retraction. In vivo, genetic deletion of Arg diminished vascular leak in the skin and lungs, in the presence of a normal immune response. Together, our data indicate that Arg is a central and non-redundant regulator of endothelial barrier integrity, which contributes to cell retraction and gap formation by increasing the dynamics of adherens junctions and cell-matrix adhesions in a Rho GTPase-dependent fashion. Therapeutic inhibition of Arg may provide a suitable strategy for the treatment of a variety of clinical conditions characterized by vascular leak.

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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.

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