Polychlorinated biphenyl quinone induces endothelial barrier dysregulation by setting the cross talk between VE-cadherin, focal adhesion, and MAPK signaling

Environmental hazardous material polychlorinated biphenyl (PCB) exposure is associated with vascular endothelial dysfunction, which may increase the risk of cardiovascular diseases and cancer metastasis. Our previous studies illustrated the cytotoxic, antiproliferative, and genotoxic effects of a synthetic, quinone-type, highly reactive metabolite of PCB, 2,3,5-trichloro-6-phenyl-[1,4]benzoquinone (PCB29-pQ). Here, we used it as the model compound to investigate its effects on vascular endothelial integrity and permeability. We demonstrated that noncytotoxic doses of PCB29-pQ induced vascular endothelial (VE)-cadherin junction disassembly by increasing the phosphorylation of VE-cadherin at Y658. We also found that focal adhesion assembly was required for PCB29-pQ-induced junction breakdown. Focal adhesion site-associated actin stress fibers may serve as holding points for cytoskeletal tension to regulate the cellular contractility. PCB29-pQ exposure promoted the association of actin stress fibers with paxillin-containing focal adhesion sites and enlarged the size/number of focal adhesions. In addition, PCB29-pQ treatment induced phosphorylation of paxillin at Y118. By using pharmacological inhibition, we further demonstrated that p38 activation was necessary for paxillin phosphorylation, whereas extracellular signal-regulated kinases-1/2 activation regulated VE-cadherin phosphorylation. In conclusion, these results indicated that PCB29-pQ stimulates endothelial hyperpermeability by mediating VE-cadherin disassembly, junction breakdown, and focal adhesion formation. Intervention strategies targeting focal adhesion and MAPK signaling could be used as therapeutic approaches for preventing adverse cardiovascular health effects induced by environmental toxicants such as PCBs.

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A Theoretical Model of Stretch-Induced Stress Fiber Remodeling

ASME 2008 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2008-193241 ◽

2008 ◽

Author(s):

Roland Kaunas

Keyword(s):

Focal Adhesion ◽

Focal Adhesions ◽

Dynamic Structure ◽

Cytoskeletal Proteins ◽

Stress Fibers ◽

Cell Contractility ◽

Maximum Stretch ◽

Tensional Homeostasis ◽

Tractional Forces ◽

Actin Stress Fibers

Cyclic stretching of endothelial cells (ECs), such as occurs in arteries during the cardiac cycle, induces ECs and their actin stress fibers to orient perpendicular to the direction of maximum stretch. This perpendicular alignment response is strengthened by increasing the magnitudes of stretch and cell contractility (1). The actin cytoskeleton is a dynamic structure that regulates cell shape changes and mechanical properties. It has been shown that actin stress fibers are ‘prestretched’ under normal, non-perturbed, conditions (2), consistent with the ideas of ‘prestress’ that have motivated tensegrity cell models (3). It has also been shown that ‘tractional forces’ generated by cells at focal adhesions tend to increase proportionately with increasing focal adhesion area, thus suggesting that cells tend to maintain constant the stress borne by a focal adhesion (4). By implication, this suggests that cells try to maintain constant the stress in actin stress fibers. Thus, it seems that cells reorganize or turnover cytoskeletal proteins and adhesion complexes so as to maintain constant a preferred mechanical state. Mizutani et al. (5) referred to this as cellular tensional homeostasis, although they did not suggest a model or theory to account for this dynamic process.

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Activation of Rho-kinase and focal adhesion kinase regulates the organization of stress fibers and focal adhesions in the central part of fibroblasts

10.7287/peerj.preprints.3297v1 ◽

2017 ◽

Author(s):

Kazuo Katoh

Keyword(s):

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Adhesion Formation ◽

Rho Kinase ◽

Focal Adhesions ◽

Fiber Formation ◽

Stress Fibers ◽

Focal Adhesion Formation ◽

Specific Regulation

Specific regulation and activation of focal adhesion kinase (FAK) are thought to be important for focal adhesion formation, and activation of Rho-kinase has been suggested to play a role in determining the effects of FAK on the formation of stress fibers and focal adhesions. To clarify the role of FAK in stress fiber formation and focal adhesion organization, we examined the formation of new stress fibers and focal adhesions by activation of Rho-kinase in FAK knockout (FAK–/–) fibroblasts. FAK–/– cells were elliptical in shape, and showed reduced numbers of stress fibers and focal adhesions in the central part of the cells along with large focal adhesions in the peripheral regions. Activation of Rho-kinase in FAK–/– cells transiently increased the actin filaments in the cell center, but these did not form typical thick stress fibers. Moreover, only plaque-like structures as the origins of newly formed focal adhesions were observed in the center of the cell. Furthermore, introduction of an exogenous GFP-labeled FAK gene into FAK–/– cells resulted in increased numbers of stress fibers and focal adhesions in the center of the cells, which showed typical fibroblast morphology. These results indicated that FAK plays an important role in the formation of stress fibers and focal adhesions as well as in regulation of cell shape and morphology with the activation of Rho-kinase.

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ARF1 Mediates Paxillin Recruitment to Focal Adhesions and Potentiates Rho-stimulated Stress Fiber Formation in Intact and Permeabilized Swiss 3T3 Fibroblasts

The Journal of Cell Biology ◽

10.1083/jcb.143.7.1981 ◽

1998 ◽

Vol 143 (7) ◽

pp. 1981-1995 ◽

Cited By ~ 108

Author(s):

J.C. Norman ◽

D. Jones ◽

S.T. Barry ◽

M.R. Holt ◽

S. Cockcroft ◽

...

Keyword(s):

Focal Adhesion ◽

Focal Adhesions ◽

Stress Fiber ◽

Fiber Formation ◽

Stress Fibers ◽

3T3 Fibroblasts ◽

Stress Fiber Formation ◽

Swiss 3T3 ◽

Swiss 3T3 Fibroblasts ◽

Actin Stress Fibers

Focal adhesion assembly and actin stress fiber formation were studied in serum-starved Swiss 3T3 fibroblasts permeabilized with streptolysin-O. Permeabilization in the presence of GTPγS stimulated rho-dependent formation of stress fibers, and the redistribution of vinculin and paxillin from a perinuclear location to focal adhesions. Addition of GTPγS at 8 min after permeabilization still induced paxillin recruitment to focal adhesion–like structures at the ends of stress fibers, but vinculin remained in the perinuclear region, indicating that the distributions of these two proteins are regulated by different mechanisms. Paxillin recruitment was largely rho-independent, but could be evoked using constitutively active Q71L ADP-ribosylation factor (ARF1), and blocked by NH2-terminally truncated Δ17ARF1. Moreover, leakage of endogenous ARF from cells was coincident with loss of GTPγS- induced redistribution of paxillin to focal adhesions, and the response was recovered by addition of ARF1. The ability of ARF1 to regulate paxillin recruitment to focal adhesions was confirmed by microinjection of Q71LARF1 and Δ17ARF1 into intact cells. Interestingly, these experiments showed that V14RhoA- induced assembly of actin stress fibers was potentiated by Q71LARF1. We conclude that rho and ARF1 activate complimentary pathways that together lead to the formation of paxillin-rich focal adhesions at the ends of prominent actin stress fibers.

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Activation of Rho-kinase and focal adhesion kinase regulates the organization of stress fibers and focal adhesions in the central part of fibroblasts

10.7287/peerj.preprints.3297 ◽

2017 ◽

Author(s):

Kazuo Katoh

Keyword(s):

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Adhesion Formation ◽

Rho Kinase ◽

Focal Adhesions ◽

Fiber Formation ◽

Stress Fibers ◽

Focal Adhesion Formation ◽

Specific Regulation

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A Focal Adhesion Filament Cross-correlation Kit for fast, automated segmentation and correlation of focal adhesions and actin stress fibers in cells

PLoS ONE ◽

10.1371/journal.pone.0250749 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0250749

Author(s):

Lara Hauke ◽

Shwetha Narasimhan ◽

Andreas Primeßnig ◽

Irina Kaverina ◽

Florian Rehfeldt

Keyword(s):

Focal Adhesion ◽

Cross Correlation ◽

Focal Adhesions ◽

Force Production ◽

Traction Force ◽

Stress Fibers ◽

Coupled Analysis ◽

Matrix Remodeling ◽

Fiber System ◽

Actin Stress Fibers

Focal adhesions (FAs) and associated actin stress fibers (SFs) form a complex mechanical system that mediates bidirectional interactions between cells and their environment. This linked network is essential for mechanosensing, force production and force transduction, thus directly governing cellular processes like polarization, migration and extracellular matrix remodeling. We introduce a tool for fast and robust coupled analysis of both FAs and SFs named the Focal Adhesion Filament Cross-correlation Kit (FAFCK). Our software can detect and record location, axes lengths, area, orientation, and aspect ratio of focal adhesion structures as well as the location, length, width and orientation of actin stress fibers. This enables users to automate analysis of the correlation of FAs and SFs and study the stress fiber system in a higher degree, pivotal to accurately evaluate transmission of mechanocellular forces between a cell and its surroundings. The FAFCK is particularly suited for unbiased and systematic quantitative analysis of FAs and SFs necessary for novel approaches of traction force microscopy that uses the additional data from the cellular side to calculate the stress distribution in the substrate. For validation and comparison with other tools, we provide datasets of cells of varying quality that are labelled by a human expert. Datasets and FAFCK are freely available as open source under the GNU General Public License.

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Protein kinase C involvement in focal adhesion formation

Journal of Cell Science ◽

10.1242/jcs.101.2.277 ◽

1992 ◽

Vol 101 (2) ◽

pp. 277-290 ◽

Cited By ~ 45

Author(s):

A. Woods ◽

J.R. Couchman

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Focal Adhesion ◽

Kinase Inhibitors ◽

Phorbol Esters ◽

Adhesion Formation ◽

Focal Adhesions ◽

Stress Fibers ◽

Kinase C ◽

Focal Adhesion Formation

Matrix molecules such as fibronectin can promote cell attachment, spreading and focal adhesion formation. Although some interactions of fibronectin with cell surface receptors have now been identified, the consequent activation of intracellular messenger systems by cell/matrix interactions have still to be elucidated. We show here that the kinase inhibitors H7 and HA1004 reduce focal adhesion and stress fiber formation in response to fibronectin in a dose-dependent manner, and that activators of protein kinase C can promote their formation under conditions where they do not normally form. Fibroblasts spread within 1h on substrata composed of fibronectin and formed focal adhesions by 3h, as monitored by interference reflection microscopy (IRM) and by labeling for talin, vinculin and integrin beta 1 subunits. In addition, stress fibers were visible. When cells were allowed to spread for 1h and then treated with kinase inhibitors H7 and HA1004 for 2h, IRM indicated a reduction in focal adhesion formation at concentrations where protein kinase C (PKC) should be inhibited. In contrast, focal adhesions formed normally at concentrations of these inhibitors where cyclic AMP- or cyclic GMP-dependent kinases should be inactivated. Inhibition of PKC, but not that of cyclic AMP- or cyclic GMP-dependent kinases, also prevented the formation of stress fibers and induced a dispersal of talin and vinculin, but not integrin beta 1 subunits, from small condensations present at 1h. Consistent with the reduction in focal adhesion formation when PKC was inhibited, activation of PKC by 30 minutes of treatment with phorbol esters induced focal adhesion formation in cells spread for 3h on substrata composed of the cell-binding (RGD-containing) fragment of fibronectin, while untreated cells or those treated with inactive phorbol esters did not form these structures.

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Mapping the dynamics of shear stress-induced structural changes in endothelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00457.2006 ◽

2007 ◽

Vol 293 (5) ◽

pp. C1616-C1626 ◽

Cited By ~ 61

Author(s):

Rosalind E. Mott ◽

Brian P. Helmke

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Structural Dynamics ◽

Focal Adhesion ◽

Actin Polymerization ◽

Lateral Displacement ◽

Focal Adhesions ◽

Stress Fibers ◽

Cell Layers ◽

Actin Stress Fibers

Hemodynamic shear stress regulates endothelial cell biochemical processes that govern cytoskeletal contractility, focal adhesion dynamics, and extracellular matrix (ECM) assembly. Since shear stress causes rapid strain focusing at discrete locations in the cytoskeleton, we hypothesized that shear stress coordinately alters structural dynamics in the cytoskeleton, focal adhesion sites, and ECM on a time scale of minutes. Using multiwavelength four-dimensional fluorescence microscopy, we measured the displacement of rhodamine-fibronectin and green fluorescent protein-labeled actin, vimentin, paxillin, and/or vinculin in aortic endothelial cells before and after onset of steady unidirectional shear stress. In the cytoskeleton, the onset of shear stress increased actin polymerization into lamellipodia, altered the angle of lateral displacement of actin stress fibers and vimentin filaments, and decreased centripetal remodeling of actin stress fibers in subconfluent and confluent cell layers. Shear stress induced the formation of new focal complexes and reduced the centripetal remodeling of focal adhesions in regions of new actin polymerization. The structural dynamics of focal adhesions and the fibronectin matrix varied with cell density. In subconfluent cell layers, shear stress onset decreased the displacement of focal adhesions and fibronectin fibrils. In confluent monolayers, the direction of fibronectin and focal adhesion displacement shifted significantly toward the downstream direction within 1 min after onset of shear stress. These spatially coordinated rapid changes in the structural dynamics of cytoskeleton, focal adhesions, and ECM are consistent with focusing of mechanical stress and/or strain near major sites of shear stress-mediated mechanotransduction.

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A Focal Adhesion Filament Cross-correlation Kit for fast, automated segmentation and correlation of focal adhesions and actin stress fibers in cells

10.1101/2021.04.14.439781 ◽

2021 ◽

Author(s):

Lara Hauke ◽

Shwetha Narasimhan ◽

Andreas Primeßnig ◽

Irina Kaverina ◽

Florian Rehfeldt

Keyword(s):

Extracellular Matrix ◽

Quantitative Analysis ◽

Focal Adhesion ◽

Cross Correlation ◽

Focal Adhesions ◽

Traction Force ◽

Stress Fibers ◽

Traction Force Microscopy ◽

Force Microscopy ◽

Actin Stress Fibers

AbstractFocal adhesions (FAs) and associated actin stress fibers (SFs) form a complex mechanical system that mediates bidirectional interactions between cells and their environment. This linked network is essential for mechanosensing, force production and force transduction, thus directly governing cellular processes like polarization, migration and extracellular matrix remodeling. We introduce a tool for fast and robust coupled analysis of both FAs and SFs named the Focal Adhesion Filament Cross-correlation Kit (FAFCK). Our software can detect and record location, axes lengths, area, orientation, and aspect ratio of focal adhesion structures as well as the location, length, width and orientation of actin stress fibers. This enables users to automate analysis of the correlation of FAs and SFs and study the stress fiber system in a higher degree, pivotal to accurately evaluate transmission of mechanocellular forces between a cell and its surroundings. The FAFCK is particularly suited for unbiased and systematic quantitative analysis of FAs and SFs necessary for novel approaches of traction force microscopy that uses the additional data from the cellular side to calculate the stress distribution in the substrate. For validation and comparison with other tools, we provide datasets of cells of varying quality that are labelled by a human expert. Datasets and FAFCK are freely available as open source under the GNU General Public License.Author summaryOur novel Focal Adhesion Filament Cross-correlation Kit (FAFCK) allows for fast, reliable, unbiased, and systematic detection of focal adhesions and actin stress fibers in cells and their mutual correlation. Detailed analysis of these structures which are both key elements in mechano-sensing and force transduction will help tremendously to improve quantitative analysis of mechanocellular experiments, key to understanding the complex interplay between cells and the extracellular matrix. In particular, sophisticated analysis methods such as model-based traction force microscopy will benefit from correlating the detailed datasets of stress fibers and focal adhesions.

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Activation of Rho-kinase and focal adhesion kinase regulates the organization of stress fibers and focal adhesions in the central part of fibroblasts

PeerJ ◽

10.7717/peerj.4063 ◽

2017 ◽

Vol 5 ◽

pp. e4063 ◽

Cited By ~ 8

Author(s):

Kazuo Katoh

Keyword(s):

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Adhesion Formation ◽

Rho Kinase ◽

Focal Adhesions ◽

Fiber Formation ◽

Stress Fibers ◽

Focal Adhesion Formation ◽

Specific Regulation

Specific regulation and activation of focal adhesion kinase (FAK) are thought to be important for focal adhesion formation, and activation of Rho-kinase has been suggested to play a role in determining the effects of FAK on the formation of stress fibers and focal adhesions. To clarify the role of FAK in stress fiber formation and focal adhesion organization, the author examined the formation of new stress fibers and focal adhesions by activation of Rho-kinase in FAK knockout (FAK–/–) fibroblasts. FAK–/–cells were elliptical in shape, and showed reduced numbers of stress fibers and focal adhesions in the central part of the cells along with large focal adhesions in the peripheral regions. Activation of Rho-kinase in FAK–/–cells transiently increased the actin filaments in the cell center, but these did not form typical thick stress fibers. Moreover, only plaque-like structures as the origins of newly formed focal adhesions were observed in the center of the cell. Furthermore, introduction of an exogenous GFP-labeled FAK gene into FAK–/–cells resulted in increased numbers of stress fibers and focal adhesions in the center of the cells, which showed typical fibroblast morphology. These results indicated that FAK plays an important role in the formation of stress fibers and focal adhesions as well as in regulation of cell shape and morphology with the activation of Rho-kinase.

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Vascular-endothelial-cadherin modulates endothelial monolayer permeability

Journal of Cell Science ◽

10.1242/jcs.112.12.1915 ◽

1999 ◽

Vol 112 (12) ◽

pp. 1915-1923 ◽

Cited By ~ 3

Author(s):

P.L. Hordijk ◽

E. Anthony ◽

F.P. Mul ◽

R. Rientsma ◽

L.C. Oomen ◽

...

Keyword(s):

Cell Adhesion ◽

Actin Cytoskeleton ◽

Adhesion Molecule ◽

Cell Adhesion Molecule ◽

Transendothelial Migration ◽

Stress Fibers ◽

Vascular Endothelial ◽

Monolayer Permeability ◽

Actin Stress Fibers ◽

Cell Cell

Vascular endothelial (VE)-cadherin is the endothelium-specific member of the cadherin family of homotypic cell adhesion molecules. VE-cadherin, but not the cell adhesion molecule platelet/endothelial cell adhesion molecule (PECAM-1), markedly colocalizes with actin stress fibers at cell-cell junctions between human umbilical vein endothelial cells. Inhibition of VE-cadherin-mediated, but not PECAM-1-mediated, adhesion induced reorganization of the actin cytoskeleton, loss of junctional VE-cadherin staining and loss of cell-cell adhesion. In functional assays, inhibition of VE-cadherin caused increased monolayer permeability and enhanced neutrophil transendothelial migration. In a complementary set of experiments, modulation of the actin cytoskeleton was found to strongly affect VE-cadherin distribution. Brief stimulation of the beta2-adrenergic receptor with isoproterenol induced a loss of actin stress fibers resulting in a linear, rather than ‘jagged’, VE-cadherin distribution. The concomitant, isoproterenol-induced, reduction in monolayer permeability was alleviated by a VE-cadherin-blocking antibody. Finally, cytoskeletal reorganization resulting from the inactivation of p21Rho caused a diffuse localization of VE-cadherin, which was accompanied by reduced cell-cell adhesion. Together, these data show that monolayer permeability and neutrophil transendothelial migration are modulated by VE-cadherin-mediated cell-cell adhesion, which is in turn controlled by the dynamics of the actin cytoskeleton.

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