Heme induces rapid endothelial barrier dysfunction via the MKK3/p38MAPK axis

Abstract Several studies demonstrate that hemolysis and free heme in circulation cause endothelial barrier dysfunction and are associated with severe pathological conditions such as acute respiratory distress syndrome, acute chest syndrome, and sepsis. However, the precise molecular mechanisms involved in the pathology of heme-induced barrier disruption remain to be elucidated. In this study, we investigated the role of free heme in the endothelial barrier integrity and mechanisms of heme-mediated intracellular signaling of human lung microvascular endothelial cells (HLMVECs). Heme, in a dose-dependent manner, induced a rapid drop in the endothelial barrier integrity of HLMVECs. An investigation into barrier proteins revealed that heme primarily affected the tight junction proteins zona occludens-1, claudin-1, and claudin-5, which were significantly reduced after heme exposure. The p38MAPK/HSP27 pathway, involved in the regulation of endothelial cytoskeleton remodeling, was also significantly altered after heme treatment, both in HLMVECs and mice. By using a knockout (KO) mouse for MKK3, a key regulator of the p38MAPK pathway, we showed that this KO effectively decreased heme-induced endothelial barrier dysfunction. Taken together, our results indicate that targeting the p38MAPK pathway may represent a crucial treatment strategy in alleviating hemolytic diseases.

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Abstract 17211: Targeting Mitogen-Activated Kinase Alleviates Free Heme-Induced Endothelial Barrier Dysfunction and Vascular Leakage in Lungs

Circulation ◽

10.1161/circ.142.suppl_3.17211 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Joel James ◽

Mathews Valuparampil Varghes ◽

Marina zemskova ◽

Olga Rafikova ◽

Ruslan Rafikov

Keyword(s):

P38 Mapk ◽

Mapk Pathway ◽

Vascular Leakage ◽

Endothelial Barrier ◽

Contributory Factor ◽

Dependent Manner ◽

Barrier Dysfunction ◽

Endothelial Barrier Dysfunction ◽

P38 Mapk Pathway ◽

Free Heme

Introduction: Several studies confirm that free heme in circulation due to hemolysis causes endothelial barrier dysfunction. We recently demonstrated that hemolysis-induced vascular leakage with barrier dysfunction was a contributory factor to the development of pulmonary hypertension (PH). However, the precise molecular mechanisms involved in the pathology of heme induced barrier disruption still remains to be elucidated. Hypothesis: Previous studies by us showed that free heme activated the p38/MAPK pathway. Therefore, we hypothesized that targeting mitogen-activated protein kinase kinase 3 (MKK3) a key regulator of this pathway would alleviate heme induced vascular leakage. Methods: Barrier dysfunction in human micro-vascular endothelial cells (HLMVEC) was monitored using noninvasive electrical impedance and immunostaining. We used an MKK3 knockout mouse model to assess the efficacy of targeting the p38/MAPK pathway. Results: We found a rapid drop in the HLMVEC barrier integrity with heme, in a dose dependent manner (p<0.05). Investigating the barrier proteins showed that heme significantly affected the tight junction proteins, zona occludens-1, claudin1, and claudin5 (p<0.05). We also found the p38MAPK/HSP27 pathway, involved in regulating the endothelial cytoskeleton remodeling, to be significantly altered with heme treatment, both in the HLMVEC and mice (p<0.05). However, heme treated mice showed no significant change in E-selectin, ICAM1 and VCAM1, indicating that the primary rapid target of heme was the p38/MAPK pathway and not the inflammatory pathways. Finally, injecting mice with heme-FITC-dextran and then following its release into the lungs demonstrated that the MKK3 KO significantly prevented heme induced vascular leakage (p<0.05). Conclusion: We demonstrate that heme induces a rapid barrier dysfunction by disruption of endothelial barrier proteins via the p38/MAPK pathway. Also, knocking out MKK3, a crucial regulator of the p38/MAPK pathway significantly decreased heme induced vascular leakage, a contributory factor to PH. Taken together, our results show that targeting the MKK3/p38MAPK axis represents a decisive treatment strategy in alleviating heme induced barrier dysfunction in cardiovascular diseases.

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The hawthorn special extract WS® 1442 protects against endothelial barrier dysfunction – elucidation of the underlying molecular mechanisms

Planta Medica ◽

10.1055/s-0030-1264291 ◽

2010 ◽

Vol 76 (12) ◽

Author(s):

R Fürst ◽

M Bubik ◽

P Bihari ◽

G Jürgenliemk ◽

H Ammer ◽

...

Keyword(s):

Molecular Mechanisms ◽

Endothelial Barrier ◽

Barrier Dysfunction ◽

Endothelial Barrier Dysfunction

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Pigment Epithelium-Derived Factor Induces Endothelial Barrier Dysfunction via p38/MAPK Phosphorylation

BioMed Research International ◽

10.1155/2015/791825 ◽

2015 ◽

Vol 2015 ◽

pp. 1-7 ◽

Cited By ~ 3

Author(s):

Ting He ◽

Liping Zhao ◽

Dongxia Zhang ◽

Qiong Zhang ◽

Jiezhi Jia ◽

...

Keyword(s):

P38 Mapk ◽

Umbilical Vein ◽

Pigment Epithelium ◽

Endothelial Barrier ◽

Dependent Manner ◽

Barrier Dysfunction ◽

Edema Formation ◽

Endothelial Barrier Dysfunction ◽

Pigment Epithelium Derived Factor

Endothelial barrier dysfunction, which is a serious problem that occurs in various inflammatory conditions, permits extravasation of serum components into the surrounding tissues, leading to edema formation and organ failure. Pigment epithelium-derived factor (PEDF), which is a major endogenous antagonist, has been implicated in diverse biological process, but its role in endothelial barrier dysfunction has not been defined. To assess the role of PEDF in the vasculature, we evaluated the effects of exogenous PEDF using human umbilical vein endothelial cells (HUVECs)in vitro. Our results demonstrated that exogenous PEDF activated p38/MAPK signalling pathway in a dose- and time-dependent manner and induced vascular hyperpermeability as measured by the markedly increased FITC-dextran leakage and the decreased transendothelial electrical resistance (TER) across the monolayer cells, which was accompanied by microtubules (MTs) disassembly and F-actin rearrangement. However, the aforementioned alterations can be arrested by the application of low concentration of p38/MAPK inhibitor SB203580. These results reveal a novel role for PEDF as a potential vasoactive substance in inducing hyperpermeability. Furthermore, our results suggest that PEDF and p38/MAPK may serve as therapeutic targets for maintaining vascular integrity.

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51 HIGH-MOLECULAR-WEIGHT POLYETHYLENE GLYCOL PROTECTS THROMBIN-INDUCED ENDOTHELIAL BARRIER DYSFUNCTION BY INDUCING ACTIN CYTOSKELETON REARRANGEMENT, WHICH RESULTS IN ROBUST ENHANCEMENT OF ENDOTHELIAL CELL BARRIER INTEGRITY.

Journal of Investigative Medicine ◽

10.2310/6650.2005.x0015.50 ◽

2006 ◽

Vol 54 (2) ◽

pp. S351.6-S352

Author(s):

E. T. Chiang ◽

S. M. Camp ◽

S. M. Dudek ◽

O. Zaborina ◽

J. C. Alverdy ◽

...

Keyword(s):

Molecular Weight ◽

Polyethylene Glycol ◽

Endothelial Cell ◽

Actin Cytoskeleton ◽

High Molecular Weight ◽

Endothelial Barrier ◽

Barrier Dysfunction ◽

Barrier Integrity ◽

Endothelial Barrier Dysfunction ◽

Cytoskeleton Rearrangement

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Molecular mechanisms in lipopolysaccharide-induced pulmonary endothelial barrier dysfunction

International Immunopharmacology ◽

10.1016/j.intimp.2015.10.010 ◽

2015 ◽

Vol 29 (2) ◽

pp. 937-946 ◽

Cited By ~ 34

Author(s):

Han Liu ◽

Xiu Yu ◽

Sulan Yu ◽

Junping Kou

Keyword(s):

Molecular Mechanisms ◽

Endothelial Barrier ◽

Barrier Dysfunction ◽

Endothelial Barrier Dysfunction

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The HSP90 Inhibitor, AUY-922, Protects and Repairs Human Lung Microvascular Endothelial Cells from Hydrochloric Acid-Induced Endothelial Barrier Dysfunction

Cells ◽

10.3390/cells10061489 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1489

Author(s):

Ruben M. L. Colunga Biancatelli ◽

Pavel Solopov ◽

Betsy Gregory ◽

John D. Catravas

Keyword(s):

Endothelial Cells ◽

Hydrochloric Acid ◽

Human Lung ◽

Hsp90 Inhibitor ◽

Endothelial Barrier ◽

Microvascular Endothelial Cells ◽

Dependent Manner ◽

Barrier Dysfunction ◽

Endothelial Barrier Dysfunction ◽

Microvascular Endothelial

Exposure to hydrochloric acid (HCl) leads acutely to asthma-like symptoms, acute respiratory distress syndrome (ARDS), including compromised alveolo-capillary barrier, and respiratory failure. To better understand the direct effects of HCl on pulmonary endothelial function, we studied the characteristics of HCl-induced endothelial barrier dysfunction in primary cultures of human lung microvascular endothelial cells (HLMVEC), defined the involved molecular pathways, and tested the potentially beneficial effects of Heat Shock Protein 90 (HSP90) inhibitors. HCl impaired barrier function in a time- and concentration-dependent manner and was associated with activation of Protein Kinase B (AKT), Ras homolog family member A (RhoA) and myosin light chain 2 (MLC2), as well as loss of plasmalemmal VE-cadherin, rearrangement of cortical actin, and appearance of inter-endothelial gaps. Pre-treatment or post-treatment of HLMVEC with AUY-922, a third-generation HSP90 inhibitor, prevented and restored HCl-induced endothelial barrier dysfunction. AUY-922 increased the expression of HSP70 and inhibited the activation (phosphorylation) of extracellular-signal regulated kinase (ERK) and AKT. AUY-922 also prevented the HCl-induced activation of RhoA and MLC2 and the internalization of plasmalemmal VE-cadherin. We conclude that, by increasing the expression of cytoprotective proteins, interfering with actomyosin contractility, and enhancing the expression of junction proteins, inhibition of HSP90 may represent a useful approach for the management of HCl-induced endothelial dysfunction and acute lung injury.

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Nox2-dependent glutathionylation of endothelial NOS leads to uncoupled superoxide production and endothelial barrier dysfunction in acute lung injury

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00063.2014 ◽

2014 ◽

Vol 307 (12) ◽

pp. L987-L997 ◽

Cited By ~ 29

Author(s):

Feng Wu ◽

William S. Szczepaniak ◽

Sruti Shiva ◽

Huanbo Liu ◽

Yinna Wang ◽

...

Keyword(s):

Lung Injury ◽

Superoxide Production ◽

Endothelial Barrier ◽

Tissue Homogenate ◽

Protein Accumulation ◽

Barrier Dysfunction ◽

Barrier Integrity ◽

Endothelial Barrier Dysfunction ◽

Enos Uncoupling

Microvascular barrier integrity is dependent on bioavailable nitric oxide (NO) produced locally by endothelial NO synthase (eNOS). Under conditions of limited substrate or cofactor availability or by enzymatic modification, eNOS may become uncoupled, producing superoxide in lieu of NO. This study was designed to investigate how eNOS-dependent superoxide production contributes to endothelial barrier dysfunction in inflammatory lung injury and its regulation. C57BL/6J mice were challenged with intratracheal LPS. Bronchoalveolar lavage fluid was analyzed for protein accumulation, and lung tissue homogenate was assayed for endothelial NOS content and function. Human lung microvascular endothelial cell (HLMVEC) monolayers were exposed to LPS in vitro, and barrier integrity and superoxide production were measured. Biopterin species were quantified, and coimmunoprecipitation (Co-IP) assays were performed to identify protein interactions with eNOS that putatively drive uncoupling. Mice exposed to LPS demonstrated eNOS-dependent increased alveolar permeability without evidence for altered canonical NO signaling. LPS-induced superoxide production and permeability in HLMVEC were inhibited by the NOS inhibitor nitro-l-arginine methyl ester, eNOS-targeted siRNA, the eNOS cofactor tetrahydrobiopterin, and superoxide dismutase. Co-IP indicated that LPS stimulated the association of eNOS with NADPH oxidase 2 (Nox2), which correlated with augmented eNOS S-glutathionylation both in vitro and in vivo. In vitro, Nox2-specific inhibition prevented LPS-induced eNOS modification and increases in both superoxide production and permeability. These data indicate that eNOS uncoupling contributes to superoxide production and barrier dysfunction in the lung microvasculature after exposure to LPS. Furthermore, the results implicate Nox2-mediated eNOS- S-glutathionylation as a mechanism underlying LPS-induced eNOS uncoupling in the lung microvasculature.

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ID: 114: AN ANTAGONIST OF LYSOPHOSPHATIDIC ACID RECEPTOR1, AM966, INCREASES PULMONARY ENDOTHELIAL PERMEABILITY THROUGH ACTIVATION OF VE-CADHERIN

Journal of Investigative Medicine ◽

10.1136/jim-2016-000120.112 ◽

2016 ◽

Vol 64 (4) ◽

pp. 965.3-966

Author(s):

J Cai ◽

J Wei ◽

AM Jacko ◽

J Zhao

Keyword(s):

Lysophosphatidic Acid ◽

Barrier Function ◽

Endothelial Permeability ◽

Heart Association ◽

Endothelial Barrier ◽

Dependent Manner ◽

Gap Formation ◽

Endothelial Barrier Function ◽

Barrier Dysfunction ◽

Barrier Integrity

BackgroundMaintenance of pulmonary endothelial barrier integrity is important for reducing severity of lung injury. VE-cadherin is a major component of cell–cell adherens junctions in endothelium. In response to inflammatory stimuli, VE-cadherin is tyrosine phosphorylated, resulting in dissociation with catenins, which links to f-actin. Lysophosphatidic acid (LPA) is a bioactive lysophospholipid, which regulates cell motility. LPA has been shown to increase lung epithelial barrier integrity, while it reduces endothelial barrier function. AM966 is an antagonist exhibiting an anti-fibrotic property. However, the effect of AM966 on pulmonary endothelial barrier integrity has not been well studied.Methods and ResultsTo investigate endothelial barrier integrity, electric cell-substrate sensing (ECIS) system was used to measure permeability in human lung microvascular endothelial cells (HLMVECs). Similar to the effect of LPA, AM966 increases permeability immediately in a dose dependent manner. To investigate the molecular mechanism by which regulates AM966-mediated reduction of endothelial barrier function, HLMVECs were treated with AM966, and then phosphorylation of myosin light chain (MLC) and VE-cadherin were determined by immunoblotting. AM966 increased phosphorylation of MLC and VE-cadherin. VE-cadherin and f-actin double immunostaining revealed that AM966 induces gap formation and f-actin stress fibers as well as dissociation between VE-cadherin and f-actin.ConclusionThis study reveals that AM966 induces lung endothelial barrier dysfunction, which is regulated by phosphorylation of VE-cadherin.This work was supported by the National Institutes of Health (R01GM115389 to J.Z.), American Heart Association 12SDG9050005 (J.Z.), American Lung Association Biomedical Research Grant RG350146 (J.Z.).

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