Hemodynamic factor influencing morphology and stress fiber expression in rat umbilical vessel endothelial cells

Placenta ◽  
1996 ◽  
Vol 17 (8) ◽  
pp. A8-A8
Author(s):  
H ISHIKAWA ◽  
R SAWA ◽  
Y YONEYAMA ◽  
S SHIN ◽  
S KEIJI ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Stress Fiber ◽  
Umbilical Vessel

Endothelial cytoskeletal reorganization in response to PAR1 stimulation is mediated by membrane rafts but not caveolae

2007 ◽  
Vol 293 (1) ◽  
pp. H366-H375 ◽  
Author(s):  
MaryEllen Carlile-Klusacek ◽  
Victor Rizzo
Keyword(s):  
Endothelial Cells ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Signaling Cascade ◽  
Membrane Rafts ◽  
Actin Stress Fiber ◽  
Caveolin 1 ◽  
Stress Fiber Formation ◽  
Mlc Phosphorylation ◽  
Actin Stress Fiber Formation

The vasoactive protease thrombin is a known activator of the protease-activated receptor-1 (PAR1) via cleavage of its NH2 terminus. PAR1 activation stimulates the RhoA/Rho kinase signaling cascade, leading to myosin light chain (MLC) phosphorylation, actin stress fiber formation, and changes in endothelial monolayer integrity. Previous studies suggest that some elements of this signaling pathway are localized to caveolin-containing cholesterol-rich membrane domains. Here we show that PAR1 and key components of the PAR-associated signaling cascade localize to membrane rafts and caveolae in bovine aortic endothelial cells (BAEC). To investigate the functional significance of this localization, BAEC were pretreated with filipin (5 μg/ml, 5 min) to ablate lipid rafts before thrombin (100 nM) or PAR agonist stimulation. We found that diphosphorylation of MLC and the actin stress fiber formation normally induced by PAR activation were attenuated after lipid raft disruption. To target caveolae specifically, we used a small interferring RNA approach to knockdown caveolin-1 expression. Thrombin-induced MLC phosphorylation and stress fiber formation were not altered in caveolin-1-depleted cells, suggesting that lipid rafts, but not necessarily caveolae, modulate thrombin-activated signaling pathways leading to alteration of the actin cytoskeleton in endothelial cells.

Regulation of the RhoA pathway in human endothelial cell spreading on type IV collagen: role of calcium influx

1999 ◽  
Vol 112 (19) ◽  
pp. 3205-3213 ◽  
Author(s):  
L. Masiero ◽  
K.A. Lapidos ◽  
I. Ambudkar ◽  
E.C. Kohn
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Focal Adhesion ◽  
Type Iv Collagen ◽  
Cell Spreading ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Type Iv ◽  
Stress Fiber Formation

We have shown that nonvoltage-operated Ca(2+) entry regulates human umbilical vein endothelial cell adhesion, migration, and proliferation on type IV collagen. We now demonstrate a requirement for Ca(2+) influx for activation of the RhoA pathway during endothelial cell spreading on type IV collagen. Reorganization of actin into stress fibers was complete when the cells where fully spread at 90 minutes. No actin organization into stress fibers was seen in endothelial cells plated on type I collagen, indicating a permissive effect of type IV collagen. CAI, a blocker of nonvoltage-operated Ca(2+) channels, prevented development of stress fiber formation in endothelial cells on type IV collagen. This permissive effect was augmented by Ca(2+) influx, as stimulated by 0. 5 microM thapsigargin or 0.1 microM ionomycin, yielding faster development of actin stress fibers. Ca(2+) influx and actin rearrangement in response to thapsigargin and ionomycin were abrogated by CAI. Activated, membrane-bound RhoA is a substrate for C3 exoenzyme which ADP-ribosylates and inactivates RhoA, preventing actin stress fiber formation. Pretreatment of endothelial cells with C3 exoenzyme prevented basal and thapsigargin-augmented stress fiber formation. While regulation of Ca(2+) influx did not alter RhoA translocation, it reduced in vitro ADP-ribosylation of RhoA (P(2)<0. 05), suggesting Ca(2+) influx is needed for RhoA activation during spreading on type IV collagen; no Ca(2+) regulated change in RhoA was seen in HUVECs spreading on type I collagen matrix. Blockade of Ca(2+) influx of HUVEC spread on type IV collagen also reduced tyrosine phosphorylation of p190Rho-GAP and blocked thapsigargin-enhanced binding of p190Rho-GAP to focal adhesion kinase. Thus, Ca(2+) influx is necessary for RhoA activation and for linkage of the RhoA/stress fiber cascade to the focal adhesion/focal adhesion kinase pathway during human umbilical vein endothelial cell spreading on type IV collagen.

scholarly journals Monocyte Adhesion and Spreading on Human Endothelial Cells Is Dependent on Rho-regulated Receptor Clustering

1999 ◽  
Vol 145 (6) ◽  
pp. 1293-1307 ◽  
Author(s):  
Beata Wójciak-Stothard ◽  
Lynn Williams ◽  
Anne J. Ridley
Keyword(s):  
Endothelial Cells ◽  
Stress Fiber ◽  
Cytochalasin D ◽  
Fiber Formation ◽  
Dominant Negative ◽  
Cross Linking ◽  
Monocyte Adhesion ◽  
Human Endothelial Cells ◽  
Receptor Clustering ◽  
Fiber Assembly

The GTPase Rho is known to mediate the assembly of integrin-containing focal adhesions and actin stress fibers. Here, we investigate the role of Rho in regulating the distribution of the monocyte-binding receptors E-selectin, ICAM-1, and VCAM-1 in human endothelial cells. Inhibition of Rho activity with C3 transferase or N19RhoA, a dominant negative RhoA mutant, reduced the adhesion of monocytes to activated endothelial cells and inhibited their spreading. Similar effects were observed after pretreatment of endothelial cells with cytochalasin D. In contrast, dominant negative Rac and Cdc42 proteins did not affect monocyte adhesion or spreading. C3 transferase and cytochalasin D did not alter the expression levels of monocyte-binding receptors on endothelial cells, but did inhibit clustering of E-selectin, ICAM-1, and VCAM-1 on the cell surface induced by monocyte adhesion or cross-linking antibodies. Similarly, N19RhoA inhibited receptor clustering. Monocyte adhesion and receptor cross-linking induced stress fiber assembly, and inhibitors of myosin light chain kinase prevented this response but did not affect receptor clustering. Finally, receptor clusters colocalized with ezrin/moesin/ radixin proteins. These results suggest that Rho is required in endothelial cells for the assembly of stable adhesions with monocytes via the clustering of monocyte-binding receptors and their association with the actin cytoskeleton, independent of stress fiber formation.

scholarly journals Cytokine-induced F-actin reorganization in endothelial cells involves RhoA activation

2009 ◽  
Vol 296 (3) ◽  
pp. F487-F495 ◽  
Author(s):  
Silvia B. Campos ◽  
Sharon L. Ashworth ◽  
Sarah Wean ◽  
Melanie Hosford ◽  
Ruben M. Sandoval ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Kidney Injury ◽  
Stress Fiber ◽  
Stress Fibers ◽  
Actin Dynamics ◽  
Nuclear Staining ◽  
Inflammatory Reactions ◽  
Rhoa Activation ◽  
Cytokine Cocktail ◽  
Cofilin Phosphorylation

Acute ischemic kidney injury results in marked increases in local and systemic cytokine levels. IL-1α, IL-6, and TNF-α orchestrate various inflammatory reactions influencing endothelial permeability by altering cell-to-cell and cell-to-extracellular matrix attachments. To explore the role of actin and the regulatory proteins RhoA and cofilin in this process, microvascular endothelial cells (MS1) were exposed to individual cytokines or a cytokine cocktail. Within minutes, a marked, time-dependent redistribution of the actin cytoskeleton occurred with the formation of long, dense F-actin basal stress fibers. The concentration of F-actin, normalized to nuclear staining, significantly increased compared with untreated cells (up 20%, P ≤ 0.05). Western blot analysis of MS1 lysates incubated with the cytokine cocktail for 4 h showed an increase in phosphorylated/inactive cofilin (up 25 ± 15%, P ≤ 0.05) and RhoA activation (up to 227 ± 26% increase, P ≤ 0.05) compared with untreated cells. Decreasing RhoA levels using small interfering RNA blocked the effect of cytokines on stress fiber organization. Treatment with Y-27632, an inhibitor of the RhoA effector p160-ROCK, decreased levels of phosphorylated cofilin and reduced stress fiber fluorescence by 22%. In cells treated with Y-27632 followed by treatment with the cytokine cocktail, stress fiber levels were similar to control cells and cofilin phosphorylation was 55% of control levels. Taken together, these studies demonstrate cytokine stimulation of RhoA, which in turn leads to cofilin phosphorylation and formation of numerous basal actin stress fibers. These results suggest cytokines signal through the Rho-ROCK pathway, but also through another pathway to affect actin dynamics.

Actin stress fiber pre-extension in human aortic endothelial cells

2008 ◽  
Vol 65 (4) ◽  
pp. 281-294 ◽  
Author(s):  
Lan Lu ◽  
Yunfeng Feng ◽  
William J. Hucker ◽  
Sara J. Oswald ◽  
Gregory D. Longmore ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Stress Fiber ◽  
Actin Stress Fiber ◽  
Aortic Endothelial Cells ◽  
Human Aortic Endothelial Cells ◽  
Aortic Endothelial

Tumor necrosis factor-α-induced TRPC1 expression amplifies store-operated Ca2+ influx and endothelial permeability

2004 ◽  
Vol 287 (6) ◽  
pp. L1303-L1313 ◽  
Author(s):  
Biman C. Paria ◽  
Stephen M. Vogel ◽  
Gias U. Ahmmed ◽  
Setara Alamgir ◽  
Jennifer Shroff ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transient Receptor Potential ◽  
Endothelial Permeability ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Actin Stress Fiber ◽  
Cationic Current ◽  
Stress Fiber Formation ◽  
Tnf Α ◽  
Actin Stress Fiber Formation

We determined the effects of TNF-α on the expression of transient receptor potential channel (TRPC) homologues in human vascular endothelial cells and the consequences of TRPC expression on the endothelial permeability response. We observed that TNF-α exposure increased TRPC1 expression without significantly altering expression of other TRPC isoforms in human pulmonary artery endothelial cells (HPAEC). Because TRPC1 belongs to the store-operated cation channel family, we measured the Ca2+ store depletion-mediated Ca2+ influx in response to thrombin exposure. We observed that thrombin-induced Ca2+ influx in TNF-α-stimulated HPAEC was twofold greater than in control cells. To address the relationship between store-operated Ca2+ influx and TRPC1 expression, we overexpressed TRPC1 by three- to fourfold in the human dermal microvascular endothelial cell line (HMEC) using the TRPC1 cDNA. Thrombin-induced store Ca2+ depletion in these cells caused approximately twofold greater increase in Ca2+ influx than in control cells. Furthermore, the inositol 1,4,5-trisphosphate-sensitive store-operated cationic current was increased greater than twofold in TRPC1-transfected cells compared with control. To address the role of Ca2+ influx via TRPC1 in signaling endothelial permeability, we measured actin-stress fiber formation and transendothelial monolayer electrical resistance (TER) in the TRPC1 cDNA-transfected HMEC and TNF-α-challenged HPAEC. Both thrombin-induced actin-stress fiber formation and a decrease in TER were augmented in TRPC1-overexpressing HMEC compared with control cells. TNF-α-induced increased TRPC1 expression in HPAEC also resulted in marked endothelial barrier dysfunction in response to thrombin. These findings indicate the expression level of TRPC1 in endothelial cells is a critical determinant of Ca2+ influx and signaling of the increase in endothelial permeability.

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