scholarly journals Rho GTPases have diverse effects on the organization of the actin filament system

2004 ◽  
Vol 377 (2) ◽  
pp. 327-337 ◽  
Author(s):  
Pontus ASPENSTRÖM ◽  
Åsa FRANSSON ◽  
Jan SARAS
Keyword(s):  
Endothelial Cells ◽  
Actin Filament ◽  
Focal Adhesion ◽  
Rho Gtpases ◽  
Actin Filaments ◽  
Rho Gtpase ◽  
Major Influence ◽  
Aortic Endothelial Cells ◽  
Filament System ◽  
Porcine Aortic Endothelial Cells

The Rho GTPases are related to the Ras proto-oncogenes and consist of 22 family members. These proteins have important roles in regulating the organization of the actin filament system, and thereby the morphogenesis of vertebrate cells as well as their ability to migrate. In an effort to compare the effects of all members of the Rho GTPase family, active Rho GTPases were transfected into porcine aortic endothelial cells and the effects on the actin filament system were monitored. Cdc42, TCL (TC10-like), Rac1–Rac3 and RhoG induced the formation of lamellipodia, whereas Cdc42, Rac1 and Rac2 also induced the formation of thick bundles of actin filaments. In contrast, transfection with TC10 or Chp resulted in the formation of focal adhesion-like structures, whereas Wrch-1 induced long and thin filopodia. Transfection with RhoA, RhoB or RhoC induced the assembly of stress fibres, whereas Rnd1–Rnd3 resulted in the loss of stress fibres, but this effect was associated with the formation of actin- and ezrin-containing dorsal microvilli. Cells expressing RhoD and Rif had extremely long and flexible filopodia. None of the RhoBTB or Miro GTPases had any major influence on the organization of the actin filament system; instead, RhoBTB1 and RhoBTB2 were present in vesicular structures, and Miro-1 and Miro-2 were present in mitochondria. Collectively, the data obtained in this study to some extent confirm earlier observations, but also allow the identification of previously undetected roles of the different members of the Rho GTPases.

Disintegration of cytoskeletal structure of actin filaments in energy-depleted endothelial cells

1993 ◽  
Vol 264 (5) ◽  
pp. H1599-H1608 ◽  
Author(s):  
W. Kuhne ◽  
M. Besselmann ◽  
T. Noll ◽  
A. Muhs ◽  
H. Watanabe ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Actin Filaments ◽  
Fold Increase ◽  
Metabolic Inhibition ◽  
Ionophore A23187 ◽  
Aortic Endothelial Cells ◽  
Monolayer Cultures ◽  
Cell Retraction ◽  
Porcine Aortic Endothelial Cells ◽  
Reversible Phase

In a previous study [Watanabe, H., W. Kuhne, R. Spahr, P. Schwartz, and H. M. Piper. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H1344-H1352, 1991] metabolic inhibition (5 mM KCN + 5 mM 2-deoxy-D-glucose, for 2 h) was found to cause disintegration of F-actin filaments, cell retraction, and augmented paracellular macromolecule permeability in monolayer cultures of porcine aortic endothelial cells after a rapid depletion of ATP stores (90% in 5 min). These changes were reversible. In the present study, the nature of this cytoskeletal disintegration was investigated. 1) Disintegration of F-actin filaments within 2-h incubation under metabolic inhibition was accompanied by appearance of F-actin clumps in the cells, but total contents of F-actin remained unaltered. 2) Cytosolic Ca2+ levels rapidly rose in metabolically inhibited cells; after 2 h a 10-fold increase was observed. 3) Presence of the Ca2+ ionophore A23187 (10 microM) mimicked the reversible effect of metabolic inhibition on F-actin filaments and monolayer permeability but not the extensive depletion of ATP stores. 4) Existence of the Ca(2+)-activatable actin-severing protein gelsolin in endothelial cells was demonstrated. The results show that during the reversible phase of endothelial energy depletion disintegration of F-actin filaments is only partial, since it is based on their fragmentation and not depolymerization. Increase in cytosolic Ca2+ levels seems to be the primary cause for the fragmentation, possibly through the activation of gelsolin.

scholarly journals Selectivity of Guanine Nucleotide Exchange Factor-mediated Cdc42 activation in primary human endothelial cells

2019 ◽  
Author(s):  
Nathalie R. Reinhard ◽  
Sanne van der Niet ◽  
Anna Chertkova ◽  
Marten Postma ◽  
Peter L. Hordijk ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Single Cell ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Actin Dynamics ◽  
Pleckstrin Homology Domain ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Dh Domain

AbstractThe Rho GTPase family is involved in actin dynamics and regulates the barrier function of the endothelium. One of the main barrier-promoting Rho GTPases is Cdc42, also known as cell division control protein 42 homolog. Currently, regulation of Cdc42-based signaling networks in endothelial cells (ECs) lack molecular details. To examine these, we focused on a subset of 15 Rho guanine nucleotide exchange factors (GEFs), which are expressed in the endothelium. By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1. A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the full-length GEFs. Our data reveal a specific GEF dependent activation profile, with most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and pRex1 and the highest selectivity for FGD1. Additionally, we generated truncated GEF constructs that comprise only the catalytic dbl homology (DH) domain or together with the adjacent pleckstrin homology domain (DHPH). The DH domain by itself did not activate Cdc42, whereas the DHPH domain of ITSN1, ITSN2 and PLEKHG1 showed activity towards Cdc42. Together, our study characterized endothelial GEFs that may activate Cdc42, which will be of great value for the field of vascular biology.Abstract FigureGraphical Abstract

Tyrosine phosphorylation of pp125FAK and paxillin in aortic endothelial cells induced by mechanical strain

1996 ◽  
Vol 271 (2) ◽  
pp. C635-C649 ◽  
Author(s):  
Y. Yano ◽  
J. Geibel ◽  
B. E. Sumpio
Keyword(s):  
Endothelial Cells ◽  
Morphological Change ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Cyclic Strain ◽  
Aortic Endothelial Cells ◽  
Adhesion Proteins ◽  
Focal Adhesion Proteins ◽  
And Migration ◽  
Aortic Endothelial

The objective of this study was to determine whether focal adhesion proteins pp125FAK (focal adhesion kinase) and paxillin are phosphorylated on tyrosine and might play a role in the morphological change and cell migration induced by strain. Bovine aortic endothelial cells (EC) were subjected to 10% average strain at 60 cycles/min. Cyclic strain increased the tyrosine phosphorylation of pp125FAK at 30 min (3.4-fold) and 4 h (5.9-fold) and the tyrosine phosphorylation of paxillin at 4 h (2.0-fold). Confocal microscopy showed that, after 4-h exposure to strain, EC began to elongate and F-actin, pp125FAK, and paxillin aligned, although they randomly distributed in static condition. Tyrosine kinase inhibitor tyrphostin A25 (100 microM) inhibited not only the tyrosine phosphorylation of pp125FAK and paxillin but also the redistribution of pp125FAK and paxillin, morphological change, and migration of EC induced by strain. These data demonstrate that cyclic strain induced tyrosine phosphorylation and reorganization of pp125FAK and paxillin and suggest that these focal adhesion proteins play a specific role in cyclic strain-induced morphological change and migration.

scholarly journals Clinopodium tomentosum (Kunth) Govaerts Leaf Extract Influences in vitro Cell Proliferation and Angiogenesis on Primary Cultures of Porcine Aortic Endothelial Cells

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Irvin Tubon ◽  
Chiara Bernardini ◽  
Fabiana Antognoni ◽  
Roberto Mandrioli ◽  
Giulia Potente ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Primary Cultures ◽  
Ethanolic Extract ◽  
Total Phenolic ◽  
Dependent Manner ◽  
Aortic Endothelial Cells ◽  
Phenolic Components ◽  
Porcine Aortic Endothelial Cells ◽  
Aortic Endothelial

Clinopodium tomentosum (Kunth) Govaerts is an endemic species in Ecuador, where it is used as an anti-inflammatory plant to treat respiratory and digestive affections. In this work, effects of a Clinopodium tomentosum ethanolic extract (CTEE), prepared from aerial parts of the plant, were investigated on vascular endothelium functions. In particularly, angiogenesis activity was evaluated, using primary cultures of porcine aortic endothelial cells (pAECs). Cells were cultured for 24 h in the presence of CTEE different concentrations (10, 25, 50, and 100 μg/ml); no viability alterations were found in the 10-50 μg/ml range, while a slight, but significant, proliferative effect was observed at the highest dose. In addition, treatment with CTEE was able to rescue LPS-induced injury in terms of cell viability. The CTEE ability to affect angiogenesis was evaluated by scratch test analysis and by an in vitro capillary-like network assay. Treatment with 25-50 μg/ml of extract caused a significant increase in pAEC’s migration and tube formation capabilities compared to untreated cells, as results from the increased master junctions’ number. On the other hand, CTEE at 100 μg/ml did not induce the same effects. Quantitative PCR data demonstrated that FLK-1 mRNA expression significantly increased at a CTEE dose of 25 μg/ml. The CTEE phytochemical composition was assessed through HPLC-DAD; rosmarinic acid among phenolic acids and hesperidin among flavonoids were found as major phenolic components. Total phenolic content and total flavonoid content assays showed that flavonoids are the most abundant class of polyphenols. The CTEE antioxidant activity was also showed by means of the DPPH and ORAC assays. Results indicate that CTEE possesses an angiogenic capacity in a dose-dependent manner; this represents an initial step in elucidating the mechanism of the therapeutic use of the plant.

scholarly journals TNF-α, IL-4, and IFN-γ Regulate Differential Expression of P- and E-Selectin Expression by Porcine Aortic Endothelial Cells

2000 ◽  
Vol 164 (6) ◽  
pp. 3309-3315 ◽  
Author(s):  
Claire J. Stocker ◽  
Katharine L. Sugars ◽  
Olivier A. Harari ◽  
R. Clive Landis ◽  
Bernard J. Morley ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Differential Expression ◽  
Aortic Endothelial Cells ◽  
Tnf Α ◽  
Ifn Γ ◽  
Porcine Aortic Endothelial Cells ◽  
Aortic Endothelial
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