scholarly journals Polarization and sprouting of endothelial cells by angiopoietin-1 require PAK2 and paxillin-dependent Cdc42 activation

2019 ◽  
Vol 30 (17) ◽  
pp. 2227-2239 ◽  
Author(s):  
Cécile Boscher ◽  
Vanda Gaonac’h-Lovejoy ◽  
Chantal Delisle ◽  
Jean-Philippe Gratton
Keyword(s):  
Endothelial Cells ◽  
Signaling Pathway ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Dependent Manner ◽  
Barrier Integrity ◽  
Signaling Complex ◽  
Migration Polarization ◽  
Leading Edges ◽  
Angiopoietin 1

Binding of angiopoietin-1 (Ang-1) to its receptor Tie2 on endothelial cells (ECs) promotes vessel barrier integrity and angiogenesis. Here, we identify PAK2 and paxillin as critical targets of Ang-1 responsible for EC migration, polarization, and sprouting. We found that Ang-1 increases PAK2-dependent paxillin phosphorylation and remodeling of focal adhesions and that PAK2 and paxillin are required for EC polarization, migration, and angiogenic sprouting in response to Ang-1. Our findings show that Ang-1 triggers Cdc42 activation at the leading edges of migrating ECs, which is dependent on PAK2 and paxillin expression. We also established that the polarity protein Par3 interacts with Cdc42 in response to Ang-1 in a PAK2- and paxillin-dependent manner. Par3 is recruited at the leading edges of migrating cells and in focal adhesion, where it forms a signaling complex with PAK2 and paxillin in response to Ang-1. These results show that Ang-1 triggers EC polarization and angiogenic sprouting through PAK2-dependent paxillin activation and remodeling of focal adhesions, which are necessary for local activation of Cdc42 and the associated polarity complex. We have shown that PAK2 controls a signaling pathway important for angiogenic sprouting that links focal adhesions to polarity signaling in ECs.

Systemic Analysis of Tyrosine Phosphorylated Proteins in Angiopoietin-1 Induced Signaling Pathway of Endothelial Cells

2007 ◽  
Vol 6 (8) ◽  
pp. 3278-3290 ◽  
Author(s):  
Young Mee Kim ◽  
Jawon Seo ◽  
Yung Hee Kim ◽  
Jaeho Jeong ◽  
Hye Joon Joo ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Signaling Pathway ◽  
Phosphorylated Proteins ◽  
Systemic Analysis ◽  
Angiopoietin 1

scholarly journals Assessment of cellular mechanisms contributing to cAMP compartmentalization in pulmonary microvascular endothelial cells

2012 ◽  
Vol 302 (6) ◽  
pp. C839-C852 ◽  
Author(s):  
Wei P. Feinstein ◽  
Bing Zhu ◽  
Silas J. Leavesley ◽  
Sarah L. Sayner ◽  
Thomas C. Rich
Keyword(s):  
Endothelial Cells ◽  
Adenylyl Cyclase ◽  
Signaling Pathway ◽  
Camp Signaling ◽  
Microvascular Endothelial Cells ◽  
Camp Production ◽  
Barrier Integrity ◽  
Pulmonary Microvascular Endothelial Cells ◽  
Camp Signaling Pathway ◽  
Microvascular Endothelial

Cyclic AMP signals encode information required to differentially regulate a wide variety of cellular responses; yet it is not well understood how information is encrypted within these signals. An emerging concept is that compartmentalization underlies specificity within the cAMP signaling pathway. This concept is based on a series of observations indicating that cAMP levels are distinct in different regions of the cell. One such observation is that cAMP production at the plasma membrane increases pulmonary microvascular endothelial barrier integrity, whereas cAMP production in the cytosol disrupts barrier integrity. To better understand how cAMP signals might be compartmentalized, we have developed mathematical models in which cellular geometry as well as total adenylyl cyclase and phosphodiesterase activities were constrained to approximate values measured in pulmonary microvascular endothelial cells. These simulations suggest that the subcellular localizations of adenylyl cyclase and phosphodiesterase activities are by themselves insufficient to generate physiologically relevant cAMP gradients. Thus, the assembly of adenylyl cyclase, phosphodiesterase, and protein kinase A onto protein scaffolds is by itself unlikely to ensure signal specificity. Rather, our simulations suggest that reductions in the effective cAMP diffusion coefficient may facilitate the formation of substantial cAMP gradients. We conclude that reductions in the effective rate of cAMP diffusion due to buffers, structural impediments, and local changes in viscosity greatly facilitate the ability of signaling complexes to impart specificity within the cAMP signaling pathway.

EGF receptor regulation of cell motility: EGF induces disassembly of focal adhesions independently of the motility-associated PLCgamma signaling pathway

1998 ◽  
Vol 111 (5) ◽  
pp. 615-624 ◽  
Author(s):  
H. Xie ◽  
M.A. Pallero ◽  
K. Gupta ◽  
P. Chang ◽  
M.F. Ware ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Motility ◽  
Map Kinase ◽  
Signaling Pathway ◽  
Focal Adhesion ◽  
Kinase Activity ◽  
Egf Receptor ◽  
Focal Adhesions ◽  
Short Term ◽  
Egfr Kinase

A current model of growth factor-induced cell motility invokes integration of diverse biophysical processes required for cell motility, including dynamic formation and disruption of cell/substratum attachments along with extension of membrane protrusions. To define how these biophysical events are actuated by biochemical signaling pathways, we investigate here whether epidermal growth factor (EGF) induces disruption of focal adhesions in fibroblasts. We find that EGF treatment of NR6 fibroblasts presenting full-length WT EGF receptors (EGFR) reduces the fraction of cells presenting focal adhesions from approximately 60% to approximately 30% within 10 minutes. The dose dependency of focal adhesion disassembly mirrors that for EGF-enhanced cell motility, being noted at 0.1 nM EGF. EGFR kinase activity is required as cells expressing two kinase-defective EGFR constructs retain their focal adhesions in the presence of EGF. The short-term (30 minutes) disassembly of focal adhesions is reflected in decreased adhesiveness of EGF-treated cells to substratum. We further examine here known motility-associated pathways to determine whether these contribute to EGF-induced effects. We have previously demonstrated that phospholipase C(gamma) (PLCgamma) activation and mobilization of gelsolin from a plasma membrane-bound state are required for EGFR-mediated cell motility. In contrast, we find here that short-term focal adhesion disassembly is induced by a signaling-restricted truncated EGFR (c'973) which fails to activate PLCgamma or mobilize gelsolin. The PLC inhibitor U73122 has no effect on this process, nor is the actin severing capacity of gelsolin required as EGF treatment reduces focal adhesions in gelsolin-devoid fibroblasts, further supporting the contention that focal adhesion disassembly is signaled by a pathway distinct from that involving PLCgamma. Because both WT and c'973 EGFR activate the erk MAP kinase pathway, we additionally explore here this signaling pathway, not previously associated with growth factor-induced cell motility. Levels of the MEK inhibitor PD98059 that block EGF-induced mitogenesis and MAP kinase phosphorylation also abrogate EGF-induced focal adhesion disassembly and cell motility. In summary, we characterize for the first time the ability of EGFR kinase activity to directly stimulate focal adhesion disassembly and cell/substratum detachment, in relation to its ability to stimulate migration. Furthermore, we propose a model of EGF-induced motogenic cell responses in which the PLCgamma pathway stimulating cell motility is distinct from the MAP kinase-dependent signaling pathway leading to disassembly and reorganization of cell-substratum adhesion.

Abstract 555: Bone Morphogenetic Protein Modulator BMPER Induces Angiogenesis via Inhibition of Thrombospondin-1 and Activation of the Fibroblast Growth Factor Signaling Pathway

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Jennifer S Esser ◽  
Susanne Rahner ◽  
Meike Deckler ◽  
Christoph Bode ◽  
Martin Moser
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Cell Activity ◽  
Dependent Manner ◽  
Fgf Signaling ◽  
Endothelial Cell Function ◽  
Angiogenic Activity ◽  
Thrombospondin 1

Introduction: Bone morphogenetic proteins (BMP) play a key role in vascular development. Previously, we have identified BMP endothelial cell precursor-derived regulator (BMPER), an extracellular BMP modulator, to increase the angiogenic activity of endothelial cells in a concentration-dependent manner. In this project we now investigate how the BMPER effect is mediated by key molecules of angiogenesis. Methods and Results: To assess the effect of BMPER on angiogenesis-related molecules we performed an angiogenesis antibody array with BMPER-stimulated human umbilical venous endothelial cells (HUVECs) and vice versa with BMPER-silenced HUVECs compared to control conditions, respectively. We detected increased protein expression of the anti-angiogenic thrombospondin-1 (TSP-1) 48 hours after siBMPER transfection and, consistently, decreased TSP-1 expression after stimulation with BMPER (60 ng/ml; 39% ± 7.3 N=4). Furthermore, the pro-angiogenic protein bFGF was increased after BMPER stimulation, which was confirmed by realtime-PCR and western blot analysis (288.8% ± 74.8 N=3). Additionally, we detected increased FGF receptor-1 protein expression (137.7% ± 0.4 N=3) as well as FGF signaling pathway activation. Next, we investigated the interaction of BMPER and the FGF signaling pathway in endothelial cell function. BMPER stimulation increased HUVEC angiogenic activity in matrigel, migration and spheroid assays and concomitant inhibition of FGF signaling by an anti-bFGF antibody effectively inhibited the pro-angiogenic BMPER effect. Silencing of BMPER decreased the expression of FGFR1 and, accordingly, stimulation of BMPER-silenced cells with bFGF showed decreased angiogenic endothelial cell activity (65%) compared to control. The angiogenic activity of bFGF was also reduced in C57BL/6_Bmper +/- mice as assessed in the matrigel plug assay. Ex vivo aortic ring assays of C57BL/6_Bmper +/- mice confirmed a specific effect for bFGF but not for VEGF. Conclusion: In summary, BMPER inhibits the expression of the anti-angiogenic TSP-1 and increased the expression as well as activation of the pro-angiogenic FGF signaling pathway, which overall lead to the promotion of angiogenesis.

HIV-1 Tat Mediated Effects on Focal Adhesion Assembly and Permeability in Brain Microvascular Endothelial Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3105-3105
Author(s):  
Shalom Avraham ◽  
Shuxian Jiang ◽  
Tae-Hee Lee ◽  
O. Prakash ◽  
Hava Karsenty Avraham
Keyword(s):  
Endothelial Cells ◽  
Focal Adhesion ◽  
Microvascular Endothelial Cells ◽  
Mutant Form ◽  
Dependent Manner ◽  
Brain Microvascular Endothelial Cells ◽  
Adhesion Sites ◽  
Permeability Changes ◽  
Microvascular Endothelial ◽  
Hiv 1

Abstract The blood-brain barrier (BBB) is a network formed mainly by brain microvascular endothelial cells. The integrity of the BBB is critical for brain function. Breakdown of the BBB is commonly seen in AIDS patients with HIV-1-associated dementia (HAD), despite the lack of productive HIV-infection of the brain endothelium. The processes by which HIV causes these pathological conditions are not well understood. Here, we characterized the molecular mechanisms by which Tat mediates its pathogenic effects in-vitro on primary human brain microvascular endothelial cells (HBMECs). Tat treatment of HBMECs stimulated cytoskeletal organization and increased focal adhesion sites as compared to control cells or cells treated with heat-inactivated Tat. Pretreatment with Tat antibodies or with the specific inhibitor SU-1498, which interferes with VEGFR-2 (Flk-1/KDR) receptor phosphorylation, blocked the ability of Tat to stimulate focal adhesion assembly and the migration of HBMECs. Focal adhesion kinase (FAK) was tyrosine-phosphorylated by Tat and found to be an important component of focal adhesion sites. Inhibition of FAK by the dominant-interfering mutant form FRNK (FAK-related non-kinase) significantly blocked HBMEC migration and disrupted focal adhesions upon Tat activation. Furthermore, HIV-Tat induced permeability changes in HBMECs in a time dependent manner. Tat also impaired BBB permeability as observed in HIV-1 Tat transgenic mice. These studies define a mechanism for HIV-1 Tat in focal adhesion complex assembly in HBMECs, via activation of FAK, leading to cytoskeletal reorganization and permeability changes.

scholarly journals Taohong Siwu-Containing Serum Enhances Angiogenesis in Rat Aortic Endothelial Cells by Regulating the VHL/HIF-1α/VEGF Signaling Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Zhi Tang ◽  
Wangyang Li ◽  
Hongzan Xie ◽  
Shengping Jiang ◽  
Yunqing Pu ◽  
...  
Keyword(s):  
Wound Healing ◽  
Endothelial Cells ◽  
Signaling Pathway ◽  
Bone Repair ◽  
Dependent Manner ◽  
Sprague Dawley ◽  
Aortic Endothelial Cells ◽  
Vegf Signaling ◽  
Vegf Signaling Pathway ◽  
Aortic Endothelial

Background. The incidence of bone fracture and bone-related diseases is increasing every year. Angiogenesis plays a vital role in fracture healing and bone repair. This study assessed the benefits of Taohong Siwu (TSW) decoction on angiogenesis in isolated rat aortic endothelial cells (RAEC) treated with TSW-containing serum. Methods. The components of TSW decoction were analyzed by liquid chromatography-mass spectrometry (LC-MS). TSW-containing serum was prepared by gavage of TSW decoction to Sprague-Dawley (SD) rats. The effects of TSW-containing serum on the viability, migration, wound healing, and angiogenesis of RAEC were detected by the MTT, transwell, wound healing, and Matrigel lumen formation assays, respectively. In addition, the effects of an HIF-1α inhibitor on TSW-containing serum-induced RAEC were also assessed. The effects of TSW-containing serum on the expression of the HIF-1α signaling pathway were evaluated by qRT-PCR and western blot analysis. Results. LC-MS revealed that TSW decoction primarily contained isomaltulose, choline, D-gluconic acid, L-pipecolic acid, hypotaurine, albiflorin, and tryptophan. TSW-containing serum significantly increased the viability, migration, wound healing, and angiogenesis of RAEC in a dose-dependent manner. Furthermore, our results demonstrated that HIF-1α and VEGF expressions were increased in the cells of TSW-containing serum groups, whereas VHL expression was decreased. The effects of TSW-containing serum were reversed by treatment with an HIF-1α inhibitor. Conclusion. These results suggested that TSW decoction enhanced angiogenesis by regulating the VHL/HIF-1α/VEGF signaling pathway.

scholarly journals Focal adhesion kinase leads paxillin in the assembly of nascent focal adhesions in lamellipodial protrusions of migrating endothelial cells

2015 ◽  
Vol 29 (S1) ◽  
Author(s):  
Ying‐Li Hu ◽  
Shaoying Lu ◽  
Kai Szeto ◽  
Jie Sun ◽  
Yingxiao Wang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Focal Adhesions

Detachment of Human Endothelial Cells from Fibrinogen Induced by Cleaved High Molecular Weight Kininogen (HKa) Is Mediated by c-Jun Amino-Terminal Kinase (JNK) Signaling Pathway.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1809-1809
Author(s):  
Yuchuan Liu ◽  
Irma M. Sainz ◽  
Robin A. Pixley ◽  
Robert W. Colman
Keyword(s):  
Endothelial Cells ◽  
Signaling Pathway ◽  
Intracellular Signaling ◽  
Kinase Inhibitor ◽  
Umbilical Vein ◽  
Src Kinase ◽  
Focal Adhesions ◽  
Serine Kinase ◽  
Rock Inhibitor ◽  
Amino Terminal

Abstract We have shown that HKa inhibits angiogenesis by inducing detachment and apoptosis of human umbilical vein endothelial cells (HUVECs) from specific extracellular matrix proteins, such as vitronectin and fibrinogen, but not collagen. To investigate which intracellular signaling pathway led to cell detachment, several specific inhibitors were selected and utilized. SP600125, a JNK inhibitor, and PP2, a Src kinase inhibitor, both produced HUVECs detachment from surfaces coated with fibrinogen while MG-132, a NF-κB inhibitor, induced apoptosis. Neither U0126, an ERK inhibitor, nor SB202190, a p38 inhibitor, detached HUVECs from fibrinogen surfaces. By Western blot analysis, we found that HKa inhibited JNK, but not Src kinase activity. Previous investigators have demonstrated Rho-associated protein kinase (ROCK) regulates the JNK pathway. We demonstrated that Y27632, a ROCK inhibitor, not only inhibited JNK activity, but also caused detachment of HUVECs. Using phospho-antibodies, we found that HKa decreased the phosphorylation of paxillin at Ser178 site, but not Tyr 118 site indicating a serine kinase. Paxillin is a multi-domain adaptor found at the interface between the plasma membrane and the actin cytoskeleton. Paxillin phosphorylation is known to be associated with the coordinate formation of focal adhesions and stress fibres. Since the Ser178 of paxillin is a substrate for JNK, while Tyr 118 is a substrate for FAK and Src, it appears that JNK is responsible. Our studies have uncovered the novel finding that HKa detached HUVECs from fibrinogen through the ROCK-JNK-paxillin (Ser178) pathway, but not Src-paxillin (Tyr118) signaling.

scholarly journals SAPK2/p38-dependent F-Actin Reorganization Regulates Early Membrane Blebbing during Stress-induced Apoptosis

1998 ◽  
Vol 143 (5) ◽  
pp. 1361-1373 ◽  
Author(s):  
Jacques Huot ◽  
François Houle ◽  
Simon Rousseau ◽  
Réna G. Deschesnes ◽  
Girish M. Shah ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Map Kinase ◽  
Dna Fragmentation ◽  
Focal Adhesion ◽  
Actin Polymerization ◽  
Survival Function ◽  
Focal Adhesions ◽  
Actin Dynamics ◽  
Membrane Blebbing ◽  
Bleb Formation

In endothelial cells, H2O2 induces the rapid formation of focal adhesion complexes at the ventral face of the cells and a major reorganization of the actin cytoskeleton into dense transcytoplasmic stress fibers. This change in actin dynamics results from the activation of the mitogen-activated protein (MAP) kinase stress-activated protein kinase-2/p38 (SAPK2/p38), which, via MAP kinase-activated protein (MAPKAP) kinase-2/3, leads to the phosphorylation of the actin polymerization modulator heat shock protein of 27 kD (HSP27). Here we show that the concomitant activation of the extracellular signal-regulated kinase (ERK) MAP kinase pathway by H2O2 accomplishes an essential survival function during this process. When the activation of ERK was blocked with PD098059, the focal adhesion complexes formed under the plasma membrane, and the actin polymerization activity led to a rapid and intense membrane blebbing. The blebs were delimited by a thin F-actin ring and contained enhanced levels of HSP27. Later, the cells displayed hallmarks of apoptosis, such as DEVD protease activities and internucleosomal DNA fragmentation. Bleb formation but not apoptosis was blocked by extremely low concentrations of the actin polymerization inhibitor cytochalasin D or by the SAPK2 inhibitor SB203580, indicating that the two processes are not in the same linear cascade. The role of HSP27 in mediating membrane blebbing was assessed in fibroblastic cells. In control fibroblasts expressing a low level of endogenous HSP27 or in fibroblasts expressing a high level of a nonphosphorylatable HSP27, H2O2 did not induce F-actin accumulation, nor did it generate membrane blebbing activity in the presence or absence of PD098059. In contrast, in fibroblasts that expressed wild-type HSP27 to a level similar to that found in endothelial cells, H2O2 induced accumulation of F-actin and caused bleb formation when the ERK pathway was inhibited. Cis-platinum, which activated SAPK2 but induced little ERK activity, also induced membrane blebbing that was dependent on the expression of HSP27. In these cells, membrane blebbing was not followed by caspase activation or DNA fragmentation. We conclude that the HSP27-dependent actin polymerization–generating activity of SAPK2 associated with a misassembly of the focal adhesions is responsible for induction of membrane blebbing by stressing agents.

scholarly journals Hypercholesterolaemic Serum Increases the Permeability of Endothelial Cells through Zonula Occludens-1 with Phosphatidylinositol 3-Kinase Signaling Pathway

2009 ◽  
Vol 2009 ◽  
pp. 1-5 ◽  
Author(s):  
Chang Bian ◽  
Geng Xu ◽  
Jianan Wang ◽  
Ji Ma ◽  
MeiXiang Xiang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Signaling Pathway ◽  
Pi3k Signaling ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
Zonula Occludens ◽  
Cultured Endothelial Cells ◽  
Pi3k Signaling Pathway ◽  
Zonula Occludens 1 ◽  
Phosphatidylinositol 3

Purpose. Hypercholesterolemia and tight junctions play important roles in atherosclerosis. But the relationship between these two factors is unclear. In the present study, we investigated whether hypercholesterolemic serum could change the permeability of endothelial cells through altering expression and/or distribution of tight junction protein zonula occludens-1 (ZO-1). Phosphatidylinositol 3-kinase (PI3K) signaling pathway was also examined.Materials and Methods. Cultured endothelial cells were treated with different concentration levels of hypercholesterolemic serum. The expression and distribution of ZO-1, the permeability of cultured cells and the involvement of PI3K signaling pathway were measured by various methods.Results. In the present study, we found that hypercholesterolemic serum could not change the expression of ZO-1 either in mRNA or protein level. However, hypercholesterolemic serum could change the distribution of ZO-1 in cultured endothelial cells, and increase the permeability with a dose-dependent manner. When PI3K specific inhibitor wortmannin was used, the effects induced by hypercholesterolemic serum could be partly reversed. The role of PI3K signaling pathway was further confirmed by PI3K activity assay.Conclusions. Our results suggested that although hypercholesterolemic serum could not change the expression of ZO-1, it could change the distribution and increase the permeability in endothelial cells through PI3K signaling pathway.

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