scholarly journals EXPRESS: Particulate Matter Disrupts Human Lung Endothelial Cell Barrier Integrity via Rho-dependent Pathways

2017 ◽  
pp. 204589321771721
Author(s):  
Ting Wang ◽  
Yuka Shimizu ◽  
Xiaomin Wu ◽  
Gabriel T. Kelly ◽  
Xiaoyan Xu ◽  
...  
Keyword(s):  
Particulate Matter ◽  
Endothelial Cell ◽  
Human Lung ◽  
Barrier Integrity

ID: 129: PARTICULATE MATTER DISRUPTS ENDOTHELIAL CELL PERMEABILITY VIA GAP JUNCTION PROTEIN

2016 ◽  
Vol 64 (4) ◽  
pp. 970.2-971
Author(s):  
X Wu ◽  
X Xu ◽  
JG Garcia ◽  
T Wang
Keyword(s):  
Signal Transduction ◽  
Particulate Matter ◽  
Endothelial Cell ◽  
Gap Junction ◽  
Cell Permeability ◽  
Mrna Levels ◽  
Barrier Integrity ◽  
Junction Protein ◽  
Gap Junction Protein ◽  
Cx43 Mrna

IntroductionParticulate matter (PM) is significantly associated with cardiopulmonary morbidity and mortality. We previously demonstrated that PM induces endothelial barrier disruption via reactive oxygen species (ROS)-dependent mechanisms. This study is focused on characterization of PM-regulated endothelial dysfunction via connexin43 (Cx43), a Gap junction protein. Gap junction is designated as intercellular channel which allows cells to communicate with each other, share nutrients, and transfer chemical or electrical signals, in turn, enables cells in a tissue to function in a coordinated manner.Methods and ResultsCx43 protein levels were evaluated by western blotting, and band density quantified using MyImageAnalysis. Real-time PCR was conducted to determine Cx43 mRNA levels. Human pulmonary artery endothelial cell (EC) barrier function was measured using the electrical cell-substrate impedance sensing (ECIS) system (Applied Biophysics) that provides a readout of transendothelial electrical resistance (TER). PM sample (0.1–0.3 µm of aerodynamic diameter) was collected (April of 2005) from the Ft. McHenry Tunnel, Baltimore, MD using a high-volume cyclone collector. PM (100 µg/ml) induced time-dependent increases in EC Cx43 mRNA levels (∼5 fold increase at 4 hr) and protein expression which was attenuated by N-acetyl-cysteine (NAC, 5 mM, 1 hr pretreatment), an ROS scavenger. Unlike Cx43, Cx37, another connexin expressed in ECs, remained unaltered by PM challenge. In addition, EC pretreatment with a Cx43 inhibitor, connexin-mimetic peptide Gap27 (500 µM, 2 hr pretreatment), significantly attenuated PM-reduced TER reduction by 45%, suggesting a central role of Cx43 in PM-induced lung EC barrier integrity disruption and signal transduction.ConclusionsOur results suggest Cx43 as a key and novel participant in PM-mediated signal transduction that results in loss of vascular barrier integrity. Cx43 may serve as a therapeutic target in PM-induced cardiopulmonary toxicities.

scholarly journals Particulate Matter Disrupts Human Lung Endothelial Barrier Integrity via ROS- and p38 MAPK–Dependent Pathways

2010 ◽  
Vol 42 (4) ◽  
pp. 442-449 ◽  
Author(s):  
Ting Wang ◽  
Eddie T. Chiang ◽  
Liliana Moreno-Vinasco ◽  
Gabriel D. Lang ◽  
Srikanth Pendyala ◽  
...  
Keyword(s):  
Particulate Matter ◽  
P38 Mapk ◽  
Human Lung ◽  
Endothelial Barrier ◽  
Barrier Integrity

scholarly journals Particulate matter disrupts human lung endothelial cell barrier integrity via Rho-dependent pathways

2017 ◽  
Vol 7 (3) ◽  
pp. 617-623 ◽  
Author(s):  
Ting Wang ◽  
Yuka Shimizu ◽  
Xiaomin Wu ◽  
Gabriel T. Kelly ◽  
Xiaoyan Xu ◽  
...  
Keyword(s):  
Particulate Matter ◽  
Endothelial Cell ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Human Lung ◽  
Rho Gtpase ◽  
Fiber Formation ◽  
Cell Periphery ◽  
Dependent Manner ◽  
Cytoskeleton Rearrangement

Increased exposure to ambient particulate matter (PM) is associated with elevated morbidity and mortality in patients with cardiopulmonary diseases and cancer. We and others have shown that PM induces lung microvascular barrier dysfunction which potentially enhances the systemic toxicity of PM. However, the mechanisms by which PM disrupts vascular endothelial integrity remain incompletely explored. We hypothesize that PM induces endothelial cell (EC) cytoskeleton rearrangement via Rho GTPase-dependent pathways to facilitate vascular hyperpermeability. Fine PM induced time-dependent activation of cytoskeletal machinery with increases in myosin light chain (MLC) phosphorylation and EC barrier disruption measured by transendothelial electrical resistance (TER), events attenuated by the Rho-dependent kinase (ROCK) inhibitor Y-27632 or the reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC). Both Y-27632 and NAC prevented PM-induced stress fiber formation and phospho-MLC accumulation in human lung ECs. PM promotes rapid accumulation of Rho-GTP. This event is attenuated by NAC or knockdown of RhoA (siRNA). Consistent with ROCK activation, PM induced phosphorylation of myosin light chain phosphatase (MYPT) at Thr850, a post-translational modification known to inhibit phosphatase activity. Furthermore, PM activates the guanine nucleotide exchange factor (GEF) for Rho, p115, with p115 translocation to the cell periphery, in a ROS-dependent manner. Together these results demonstrate that fine PM induces EC cytoskeleton rearrangement via Rho-dependent pathways that are dependent upon the generation of oxidative stress. As the disruption of vascular integrity further contributes to cardiopulmonary physiologic derangements, these findings provide pharmacologic targets for prevention of PM-induced cardiopulmonary toxicity.

scholarly journals Comparative Toxic Effects of Manufactured Nanoparticles and Atmospheric Particulate Matter in Human Lung Epithelial Cells

2020 ◽  
Vol 18 (1) ◽  
pp. 22
Author(s):  
Yun Wu ◽  
Mei Wang ◽  
Shaojuan Luo ◽  
Yunfeng Gu ◽  
Dongyang Nie ◽  
...  
Keyword(s):  
Particulate Matter ◽  
Human Lung ◽  
A549 Cells ◽  
Lung Epithelial Cells ◽  
Atmospheric Particulate Matter ◽  
Toxic Effects ◽  
Ros Generation ◽  
Atmospheric Particulate ◽  
Significant Difference ◽  
Lung Epithelial

Although nanoparticles (NPs) have been used as simplified atmospheric particulate matter (PM) models, little experimental evidence is available to support such simulations. In this study, we comparatively assessed the toxic effects of PM and typical NPs (four carbonaceous NPs with different morphologies, metal NPs of Fe, Al, and Ti, as well as SiO2 NPs) on human lung epithelial A549 cells. The EC50 value of PM evaluated by cell viability assay was 148.7 μg/mL, closest to that of SiO2 NPs, between the values of carbonaceous NPs and metal NPs. All particles caused varying degrees of reactive oxygen species (ROS) generation and adenosine triphosphate (ATP) suppression. TiO2 NPs showed similar performance with PM in inducing ROS production (p < 0.05). Small variations between two carbonaceous NPs (graphene oxides and graphenes) and PM were also observed at 50 μg/mL. Similarly, there was no significant difference in ATP inhibition between carbonaceous NPs and PM, while markedly different effects were caused by SiO2 NP and TiO2 NP exposure. Our results indicated that carbonaceous NPs could be served as potential surrogates for urban PM. The identification of PM model may help us further explore the specific roles and mechanisms of various components in PM.

scholarly journals Rap1 Is Involved in Angiopoietin-1-Induced Cell-Cell Junction Stabilization and Endothelial Cell Sprouting

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 155
Author(s):  
Vanda Gaonac’h-Lovejoy ◽  
Cécile Boscher ◽  
Chantal Delisle ◽  
Jean-Philippe Gratton
Keyword(s):  
Endothelial Cell ◽  
Endothelial Permeability ◽  
Intercellular Junctions ◽  
Small Gtpase ◽  
Endothelial Barrier ◽  
Cell Junction ◽  
Barrier Integrity ◽  
Rap1 Activation ◽  
Angiopoietin 1 ◽  
Cell Cell

Angiopoietin-1 (Ang-1) is an important proangiogenic factor also involved in the maintenance of endothelial-barrier integrity. The small GTPase Rap1 is involved in the regulation of adherens junctions through VE-cadherin-mediated adhesion, and in endothelial permeability. While many studies established that Rap1 activation is critical for endothelial cell–cell adhesions, its roles in the antipermeability effects of Ang-1 are ill-defined. Thus, we determined the contribution of Rap1 to Ang-1-stimulated angiogenic effects on endothelial cells (ECs). We found that Rap1 is activated following Ang-1 stimulation and is required for the antipermeability effects of Ang-1 on EC monolayers. Our results also revealed that Rap1 is necessary for EC sprouting stimulated by Ang-1 but had no significant effect on Ang-1-induced EC migration and adhesion. In contrast, downregulation of VE-cadherin markedly increased the adhesiveness of ECs to the substratum, which resulted in inhibition of Ang-1-stimulated migration. These results revealed that Rap1 is central to the effects of Ang-1 at intercellular junctions of ECs, whereas VE-cadherin is also involved in the adhesion of ECs to the extracellular matrix.

scholarly journals Myosin light chain kinase (MYLK) coding polymorphisms modulate human lung endothelial cell barrier responses via altered tyrosine phosphorylation, spatial localization, and lamellipodial protrusions

2018 ◽  
Vol 8 (2) ◽  
pp. 204589401876417 ◽  
Author(s):  
Ting Wang ◽  
Mary E. Brown ◽  
Gabriel T. Kelly ◽  
Sara M. Camp ◽  
Joseph B. Mascarenhas ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Tyrosine Phosphorylation ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Human Lung ◽  
Disease Risk ◽  
Spatial Localization ◽  
Cell Periphery ◽  
Nucleotide Polymorphisms

Sphingosine 1-phosphate (S1P) is a potent bioactive endogenous lipid that signals a rearrangement of the actin cytoskeleton via the regulation of non-muscle myosin light chain kinase isoform (nmMLCK). S1P induces critical nmMLCK Y464 and Y471 phosphorylation resulting in translocation of nmMLCK to the periphery where spatially-directed increases in myosin light chain (MLC) phosphorylation and tension result in lamellipodia protrusion, increased cell-cell adhesion, and enhanced vascular barrier integrity. MYLK, the gene encoding nmMLCK, is a known candidate gene in lung inflammatory diseases, with coding genetic variants (Pro21His, Ser147Pro, Val261Ala) that confer risk for inflammatory lung injury and influence disease severity. The functional mechanisms by which these MYLK coding single nucleotide polymorphisms (SNPs) affect biologic processes to increase disease risk and severity remain elusive. In the current study, we utilized quantifiable cell immunofluorescence assays to determine the influence of MYLK coding SNPs on S1P-mediated nmMLCK phosphorylation and translocation to the human lung endothelial cell (EC) periphery . These disease-associated MYLK variants result in reduced levels of S1P-induced Y464 phosphorylation, a key site for nmMLCK enzymatic regulation and activation. Reduced Y464 phosphorylation resulted in attenuated nmMLCK protein translocation to the cell periphery. We further conducted EC kymographic assays which confirmed that lamellipodial protrusion in response to S1P challenge was retarded by expression of a MYLK transgene harboring the three MYLK coding SNPs. These data suggest that ARDS/severe asthma-associated MYLK SNPs functionally influence vascular barrier-regulatory cytoskeletal responses via direct alterations in the levels of nmMLCK tyrosine phosphorylation, spatial localization, and lamellipodial protrusions.

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