scholarly journals Activation of mtor Mediates Hyperglycemia-Induced Renal Glomerular Endothelial Hyperpermeability via the RhoA/ROCK/pmlc Signaling Pathway

2021 ◽  
Author(s):  
Jianhui Chen ◽  
Xianfan Li ◽  
Xiaolin Chen ◽  
Zengpu Yu
Keyword(s):  
Fluorescence Probe ◽  
Endothelial Permeability ◽  
Barrier Dysfunction ◽  
Actin Reorganization ◽  
Barrier Disruption ◽  
Rhoa Activity ◽  
Filtration Barrier ◽  
Activation Assay ◽  
Mlc Phosphorylation

Abstract OBJECTIVE: Hyperglycemia is associated with albuminuria and renal glomerular endothelial dysfunction in patients with diabetic nephropathy. The mTOR and RhoA/ROCK signaling pathways are involved in glomerular filtration barrier (GFB) regulation, but their role in high glucose (HG)-induced GFB dysfunction in human renal glomerular endothelial cells (HRGECs) has not been investigated. This study aimed to investigate the mechanisms of HG-induced GFB dysfunction in vitro.MATERIALS AND METHODS: HRGECs were cultured in vitro and exposed to HG. The horseradish peroxidase–albumin leakage and transendothelial electrical resistance of the endothelial monolayer were measured after HG treatment with or without rapamycin preincubation. A fluorescence probe was used to study the distribution of F-actin reorganization. The phosphorylation levels of myosin light chain (MLC) and mTOR were measured via western blotting. RhoA activity was evaluated via GTPase activation assay. The effects of blocking mTOR or the RhoA/ROCK pathway on endothelial permeability and MLC phosphorylation under HG conditions were observed.RESULTS: HG exposure induced F-actin reorganization and increased MLC phosphorylation, leading to EC barrier disruption. This effect was attenuated by treatment with rapamycin or Y-27632. Phospho-MLC (pMLC) activation in HRGECs was mediated by RhoA/ROCK signaling. mTOR and RhoA/ROCK inhibition or knockdown attenuated pMLC activation, F-actin reorganization and barrier disruption that occurred in response to HG exposure.CONCLUSIONS: Our results revealed that HG stimulation upregulated RhoA expression and activity through an mTOR-dependent pathway, leading to MLC-mediated endothelial cell cytoskeleton rearrangement and glomerular endothelial barrier dysfunction.

scholarly journals Activation of mTOR mediates hyperglycemia-induced renal glomerular endothelial hyperpermeability via the RhoA/ROCK/pMLC signaling pathway

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Xiaolin Chen ◽  
Jianhui Chen ◽  
Xianfan Li ◽  
Zengpu Yu
Keyword(s):  
Fluorescence Probe ◽  
Endothelial Permeability ◽  
Barrier Dysfunction ◽  
Actin Reorganization ◽  
Barrier Disruption ◽  
Rhoa Activity ◽  
Filtration Barrier ◽  
Activation Assay ◽  
Mlc Phosphorylation

Abstract Objective Hyperglycemia is associated with albuminuria and renal glomerular endothelial dysfunction in patients with diabetic nephropathy. The mTOR and RhoA/ROCK signaling pathways are involved in glomerular filtration barrier (GFB) regulation, but their role in high glucose (HG)-induced GFB dysfunction in human renal glomerular endothelial cells (HRGECs) has not been investigated. This study aimed to investigate the mechanisms of HG-induced GFB dysfunction in vitro. Materials and methods HRGECs were cultured in vitro and exposed to HG. The horseradish peroxidase–albumin leakage and transendothelial electrical resistance of the endothelial monolayer were measured after HG treatment with or without rapamycin preincubation. A fluorescence probe was used to study the distribution of F-actin reorganization. The phosphorylation levels of myosin light chain (MLC) and mTOR were measured via western blotting. RhoA activity was evaluated via GTPase activation assay. The effects of blocking mTOR or the RhoA/ROCK pathway on endothelial permeability and MLC phosphorylation under HG conditions were observed. Results HG exposure induced F-actin reorganization and increased MLC phosphorylation, leading to EC barrier disruption. This effect was attenuated by treatment with rapamycin or Y-27632. Phospho-MLC (pMLC) activation in HRGECs was mediated by RhoA/ROCK signaling. mTOR and RhoA/ROCK inhibition or knockdown attenuated pMLC activation, F-actin reorganization and barrier disruption that occurred in response to HG exposure. Conclusions Our results revealed that HG stimulation upregulated RhoA expression and activity through an mTOR-dependent pathway, leading to MLC-mediated endothelial cell cytoskeleton rearrangement and glomerular endothelial barrier dysfunction.

scholarly journals Pectin limits epithelial barrier disruption by Citrobacter rodentium through anti-microbial effects

2021 ◽  
Author(s):  
M. Beukema ◽  
K. Ishisono ◽  
J. de Waard ◽  
M. M. Faas ◽  
P. de Vos ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Epithelial Barrier ◽  
Citrobacter Rodentium ◽  
Barrier Dysfunction ◽  
Antimicrobial Effects ◽  
Barrier Disruption ◽  
Microbial Effects

Pectins inhibit the growth of C. rodentium in vitro, preventing attachment of C. rodentium to CMT93 epithelial cells. Through these antimicrobial effects, pectins protect the epithelium from C. rodentium-induced barrier dysfunction and damage.

scholarly journals Effects of prostaglandin lipid mediators on agonist-induced lung endothelial permeability and inflammation

2017 ◽  
Vol 313 (4) ◽  
pp. L710-L721 ◽  
Author(s):  
Yunbo Ke ◽  
Olga V. Oskolkova ◽  
Nicolene Sarich ◽  
Yufeng Tian ◽  
Albert Sitikov ◽  
...  
Keyword(s):  
Protective Effect ◽  
Vascular Endothelial Cells ◽  
Biological Activities ◽  
Cell Monolayer ◽  
Endothelial Permeability ◽  
In Vivo Model ◽  
Barrier Dysfunction ◽  
Anti Inflammatory

Prostaglandins (PG), the products of cyclooxygenase-mediated conversion of arachidonic acid, become upregulated in many situations including allergic response, inflammation, and injury, and exhibit a variety of biological activities. Previous studies described barrier-enhancing and anti-inflammatory effects of PGE2 and PGI2 on vascular endothelial cells (EC). Yet, the effects of other PG members on EC barrier and inflammatory activation have not been systematically analyzed. This study compared effects of PGE2, PGI2, PGF2α, PGA2, PGJ2, and PGD2 on human pulmonary EC. EC permeability was assessed by measurements of transendothelial electrical resistance and cell monolayer permeability for FITC-labeled tracer. Anti-inflammatory effects of PGs were evaluated by analysis of expression of adhesion molecule ICAM1 and secretion of soluble ICAM1 and cytokines by EC. PGE2, PGI2, and PGA2 exhibited the most potent barrier-enhancing effects and most efficient attenuation of thrombin-induced EC permeability and contractile response, whereas PGI2 effectively suppressed thrombin-induced permeability but was less efficient in the attenuation of prolonged EC hyperpermeability caused by interleukin-6 or bacterial wall lipopolysaccharide, LPS. PGD2 showed a modest protective effect on the EC inflammatory response, whereas PGF2α and PGJ2 were without effect on agonist-induced EC barrier dysfunction. In vivo, PGE2, PGI2, and PGA2 attenuated LPS-induced lung inflammation, whereas PGF2α and PGJ2 were without effect. Interestingly, PGD2 exhibited a protective effect in the in vivo model of LPS-induced lung injury. This study provides a comprehensive analysis of barrier-protective and anti-inflammatory effects of different prostaglandins on lung EC in vitro and in vivo and identifies PGE2, PGI2, and PGA2 as prostaglandins with the most potent protective properties.

scholarly journals hnRNPA2/B1 Ameliorates LPS-Induced Endothelial Injury through NF-κB Pathway and VE-Cadherin/β-Catenin Signaling Modulation In Vitro

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Yi Chen ◽  
Dan Tang ◽  
Linjie Zhu ◽  
Tianjie Yuan ◽  
Yingfu Jiao ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Inflammatory Responses ◽  
Umbilical Vein ◽  
Cell Metabolism ◽  
Endothelial Permeability ◽  
Underlying Mechanism ◽  
Endothelial Injury ◽  
Inflammatory Factors ◽  
Barrier Dysfunction

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) is a protein involved in the regulation of RNA processing, cell metabolism, migration, proliferation, and apoptosis. However, the effect of hnRNPA2/B1 on injured endothelial cells (ECs) remains unclear. We investigated the effect of hnRNPA2/B1 on lipopolysaccharide- (LPS-) induced vascular endothelial injury in human umbilical vein endothelial cells (HUVECs) and the underlying mechanisms. LPS was used to induce EC injury, and the roles of hnRNPA2/B1 in EC barrier dysfunction and inflammatory responses were measured by testing endothelial permeability and the expression of inflammatory factors after the suppression and overexpression of hnRNPA2/B1. To explore the underlying mechanism by which hnRNPA2/B1 regulates endothelial injury, we studied the VE-cadherin/β-catenin pathway and NF-κB activation in HUVECs. The results showed that hnRNPA2/B1 was elevated in LPS-stimulated HUVECs. Moreover, knockdown of hnRNPA2/B1 aggravated endothelial injury by increasing EC permeability and promoting the secretion of the inflammatory cytokines TNF-α, IL-1β, and IL-6. Overexpression of hnRNPA2/B1 can reduce the permeability and inflammatory response of HUVEC stimulated by LPS in vitro, while increasing the expression of VE-Cadherin and β-catenin. Furthermore, the suppression of hnRNPA2/B1 increased the LPS-induced NF-κB activation and reduced the VE-cadherin/β-catenin pathway. Taken together, these results suggest that hnRNPA2/B1 can regulate LPS-induced EC damage through regulating the NF-κB and VE-cadherin/β-catenin pathways.

Abstract 45: Endothelial Aryl Hydrocarbon Receptor Nucleatranslator (Arnt) is a Novel Regulator of Cardiac Endothelial Barrier Integrity in Diabetes

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Maura Knapp ◽  
Mei Zheng ◽  
Nikola Sladojevic ◽  
Qiong Zhao ◽  
Konstaintin G Birukov ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Barrier Function ◽  
Endothelial Permeability ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Barrier Dysfunction ◽  
Endothelial Barrier Dysfunction ◽  
Aryl Hydrocarbon ◽  
Underlying Mechanisms

Background: Diabetes leads to endothelial barrier dysfunction and altered endothelial permeability, which results in increased cardiovascular risk. ARNT, also known as HIF-1β, a transcription factor that functions as a master regulator of glucose homeostasis, has been implicated in diabetes. Endothelial-specific ARNT deletion (ArntΔEC) in mice is embryonically lethal, with hemorrhage occurring in the heart during the embryonic stage. However, the particular role of endothelial ARNT(ecARNT) in diabetes is largely unknown. We have found a significant decrease in ARNT expression in both diabetic rodent endothelial cells and diabetic human hearts. We hypothesize that a loss of ecARNT mediates endothelial barrier dysfunction during diabetes. Methods and Results: We generated inducible endothelial specific ARNT knockout mice (ecARNT-/-) by crossing mice with loxP sequences flanking exon 6 of ARNT with Cre ERT2 mice under the VE-cadherin promoter. A 90% deletion of ecARNT was achieved following two weeks of oral tamoxifen administration. ecARNT-/- mice exhibit severe blood vessel leakage, which is restricted to the heart, suggesting a distinct function for ecARNT in different tissues. Cardiomyopathy is evident 6 months after ARNT deletion. In vitro , trans-endothelial electrical resistance (TER) and transwell assays have confirmed endothelial barrier disruption in cardiac microvascular endothelial cells (CMEC) isolated from both ecARNT-/- hearts and diabetic (DB/DB) mouse hearts. To determine the underlying mechanisms by which ARNT may regulate endothelial barrier function, we performed DNA sequencing on CMEC isolated from control, ecARNT-/-, and DB/DB mice. Data suggest a significant increase in TNFa signaling, including ELAM-1 and ICAM-1 in CMEC isolated from ecARNT-/- CMEC and diabetic CMEC. Moreover, use of anti-TNFa antibody rescues endothelial barrier dysfunction in CMEC isolated from ecARNT-/- mice. Taken together, these results suggest that a reduction in ecARNT during diabetes may mediate endothelial barrier dysfunction through a TNFa signaling pathway. Conclusion: ecARNT is a critical mediator of endothelial barrier function and could potentially serve as a therapeutic target for diabetic cardiovascular diseases.

Proplatelet Formation Is Regulated by the Rho/Rock Pathway.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3147-3147 ◽  
Author(s):  
Yunhua Chang ◽  
Frederic Aurade ◽  
Frederic Larbret ◽  
Jean Pierre Le Couedic ◽  
Laurence Momeux ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Actin Dynamics ◽  
Rock Inhibitor ◽  
Actin Reorganization ◽  
Human Cd34 ◽  
Myosin Light Chain 2 ◽  
Proplatelet Formation ◽  
Cytoskeleton Dynamics ◽  
Mlc Phosphorylation

Abstract Megakaryopoiesis is a highly specialized cellular process which sustains platelet production. At the end of megakaryopoiesis, megakaryocyte (MKs) fragments into platelets via long and thin cytoplasmic extensions called proplatelets. Proplatelet formation (PPF) is associated essentially with cytoskeleton changes, including actin dynamics. The Rho/Rock pathway is a well characterized regulator of the actin reorganization. In the present study, we have tried to understand the precise role of the Rho/Rock pathway in PPF from human CD34+ derived MKs. Our results show that Rho is expressed in MKs and that its expression and activity remain stable during megakaryopoiesis. Overexpression of a RhoA dominant negatif (RhoA N19) in MKs leads to an increase in PPF. Conversely overexpression of a RhoA spontaneous active (RhoA V14) in MKs leads to a decrease in PPF. These results indicate that Rho activation could inhibit PPF in vitro. It is known that Rho/ROCK promotes actin cytoskeleton dynamics by regulating myosin light chain 2 (MLC2) phosphorylation. To demonstrate that Rho/Rock inhibits PPF through MLC2 phosphorylation, we added MLC kinase inhibitor (P18), Rho inhibitor (TatC3) and ROCK inhibitor (Y27362) in MKs culture just before PPF. Western blot analysis shows that MLC2 phosphorylation was inhibited by these 3 compounds, in contrast, PPF was significantly increased. Moreover, the platelet produced have an identical size and ultrastructure as control platelets and could be normally activated. These results suggest that Rho/ROCK could inhibit PPF through MLC phosphorylation during megakaryopoiesis.

scholarly journals TLR4 activation of TRPC6-dependent calcium signaling mediates endotoxin-induced lung vascular permeability and inflammation

2012 ◽  
Vol 209 (11) ◽  
pp. 1953-1968 ◽  
Author(s):  
Mohammad Tauseef ◽  
Nebojsa Knezevic ◽  
Koteswara R. Chava ◽  
Monica Smith ◽  
Sukriti Sukriti ◽  
...  
Keyword(s):  
Vascular Permeability ◽  
Transient Receptor Potential ◽  
Endothelial Permeability ◽  
Receptor Potential ◽  
Trpc Channels ◽  
Dependent Manner ◽  
Barrier Dysfunction ◽  
Barrier Disruption ◽  
Transient Receptor ◽  
Lung Vascular Permeability

Lung vascular endothelial barrier disruption and the accompanying inflammation are primary pathogenic features of acute lung injury (ALI); however, the basis for the development of both remains unclear. Studies have shown that activation of transient receptor potential canonical (TRPC) channels induces Ca2+ entry, which is essential for increased endothelial permeability. Here, we addressed the role of Toll-like receptor 4 (TLR4) intersection with TRPC6-dependent Ca2+ signaling in endothelial cells (ECs) in mediating lung vascular leakage and inflammation. We find that the endotoxin (lipopolysaccharide; LPS) induces Ca2+ entry in ECs in a TLR4-dependent manner. Moreover, deletion of TRPC6 renders mice resistant to endotoxin-induced barrier dysfunction and inflammation, and protects against sepsis-induced lethality. TRPC6 induces Ca2+ entry in ECs, which is secondary to the generation of diacylglycerol (DAG) induced by LPS. Ca2+ entry mediated by TRPC6, in turn, activates the nonmuscle myosin light chain kinase (MYLK), which not only increases lung vascular permeability but also serves as a scaffold to promote the interaction of myeloid differentiation factor 88 and IL-1R–associated kinase 4, which are required for NF-κB activation and lung inflammation. Our findings suggest that TRPC6-dependent Ca2+ entry into ECs, secondary to TLR4-induced DAG generation, participates in mediating both lung vascular barrier disruption and inflammation induced by endotoxin.

Shock-induced stress induces loss of microvascular endothelial Tie2 in the kidney which is not associated with reduced glomerular barrier function

2009 ◽  
Vol 297 (2) ◽  
pp. F272-F281 ◽  
Author(s):  
Matijs van Meurs ◽  
Neng F. Kurniati ◽  
Francis M. Wulfert ◽  
Sigridur A. Asgeirsdottir ◽  
Inge A. de Graaf ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Barrier Function ◽  
Human Kidney ◽  
Mrna Levels ◽  
Barrier Dysfunction ◽  
Molecular Control ◽  
Filtration Barrier ◽  
Glomerular Barrier

Both hemorrhagic shock and endotoxemia induce a pronounced vascular activation in the kidney which coincides with albuminuria and glomerular barrier dysfunction. We hypothesized that changes in Tie2, a vascular restricted receptor tyrosine kinase shown to control microvascular integrity and endothelial inflammation, underlie this loss of glomerular barrier function. In healthy murine and human kidney, Tie2 is heterogeneously expressed in all microvascular beds, although to different extents. In mice subjected to hemorrhagic and septic shock, Tie2 mRNA and protein were rapidly, and temporarily, lost from the renal microvasculature, and normalized within 24 h after initiation of the shock insult. The loss of Tie2 protein could not be attributed to shedding as both in mice and healthy volunteers subjected to endotoxemia, sTie2 levels in the systemic circulation did not change. In an attempt to identify the molecular control of Tie2, we activated glomerular endothelial cell cultures and human kidney slices in vitro with LPS or TNF-α, but did not observe a change in Tie2 mRNA levels. In parallel to the loss of Tie2 in vivo, an overt influx of neutrophils in the glomerular compartment, which coincided with proteinuria, was seen. As neutrophil-endothelial cell interactions may play a role in endothelial adaptation to shock, and these effects cannot be mimicked in vitro, we depleted neutrophils before shock induction. While this neutrophil depletion abolished proteinuria, Tie2 was not rescued, implying that Tie2 may not be a major factor controlling maintenance of the glomerular filtration barrier in this model.

scholarly journals Phosphorylation of GTP dissociation inhibitor by PKA negatively regulates RhoA

2008 ◽  
Vol 295 (5) ◽  
pp. C1161-C1168 ◽  
Author(s):  
Jing Qiao ◽  
Oksana Holian ◽  
Bao-Shiang Lee ◽  
Fei Huang ◽  
Jihang Zhang ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Strong Support ◽  
Endothelial Permeability ◽  
Kinase Assay ◽  
Protective Mechanism ◽  
Rho Proteins ◽  
Inflammatory Injury ◽  
Rhoa Activity ◽  
Serum Response

The cAMP-PKA cascade is a recognized signaling pathway important in inhibition of inflammatory injury events such as endothelial permeability and leucocyte trafficking, and a critical target of regulation is believed to be inhibition of Rho proteins. Here, we hypothesize that PKA directly phosphorylates GTP dissociation inhibitor (GDI) to negatively regulate Rho activity. Amino acid analysis of GDIα showed two potential protein kinase A (PKA) phosphorylation motifs, Ser174 and Thr182. Using in vitro kinase assay and mass spectrometry, we found that the purified PKA catalytic subunit phosphorylated GDIα-GST fusion protein and PKA motif-containing GDIα peptide at Ser174, but not Thr182. Transfection of COS-7 cells with mutated full-length GDIα at Ser174 to Ala174 (GDIα-Ser174A) abrogated the ability of cAMP to phosphorylate GDIα. However, mutation of Thr182 to Ala182 (GDIα-Thr182A) did not abrogate, and cAMP increased phosphorylation of GDIα to a similar extent as wild-type GDIα transfectants. The mutant GDIα-Ser174A, but not GDIα-Thr182A, was unable to prevent cAMP-mediated inhibition of Rho-dependent serum-response element reporter activity. Furthermore, the mutant GDIα-Ser174A was unable to prevent the thrombin-induced RhoA activation. Coprecipitation studies indicated that neither mutation of the PKA consensus sites nor phosphorylation alter GDIα binding with RhoA, suggesting that phosphorylation of Ser174 regulated preformed GDIα-RhoA complexes. The findings provide strong support that the selective phosphorylation at Ser174 by PKA is a signaling pathway in the negative regulation of RhoA activity and therefore could be a potential protective mechanism for inflammatory injury.

scholarly journals c-Abl mediated tyrosine phosphorylation of paxillin regulates LPS-induced endothelial dysfunction and lung injury

2015 ◽  
Vol 308 (10) ◽  
pp. L1025-L1038 ◽  
Author(s):  
Panfeng Fu ◽  
Peter V. Usatyuk ◽  
Abhishek Lele ◽  
Anantha Harijith ◽  
Carol C. Gregorio ◽  
...  
Keyword(s):  
Lung Injury ◽  
Tyrosine Phosphorylation ◽  
Endothelial Permeability ◽  
Endothelial Barrier ◽  
Barrier Dysfunction ◽  
Pulmonary Vascular Permeability ◽  
Endothelial Barrier Dysfunction ◽  
Lps Challenge

Paxillin is phosphorylated at multiple residues; however, the role of tyrosine phosphorylation of paxillin in endothelial barrier dysfunction and acute lung injury (ALI) remains unclear. We used siRNA and site-specific nonphosphorylable mutants of paxillin to abrogate the function of paxillin to determine its role in lung endothelial permeability and ALI. In vitro, lipopolysaccharide (LPS) challenge of human lung microvascular endothelial cells (HLMVECs) resulted in enhanced tyrosine phosphorylation of paxillin at Y31 and Y118 with no significant change in Y181 and significant barrier dysfunction. Knockdown of paxillin with siRNA attenuated LPS-induced endothelial barrier dysfunction and destabilization of VE-cadherin. LPS-induced paxillin phosphorylation at Y31 and Y118 was mediated by c-Abl tyrosine kinase, but not by Src and focal adhesion kinase. c-Abl siRNA significantly reduced LPS-induced endothelial barrier dysfunction. Transfection of HLMVECs with paxillin Y31F, Y118F, and Y31/118F double mutants mitigated LPS-induced barrier dysfunction and VE-cadherin destabilization. In vivo, the c-Abl inhibitor AG957 attenuated LPS-induced pulmonary permeability in mice. Together, these results suggest that c-Abl mediated tyrosine phosphorylation of paxillin at Y31 and Y118 regulates LPS-mediated pulmonary vascular permeability and injury.

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