Rac Regulates Endothelial Morphogenesis and Capillary Assembly

Endothelial cells undergo branching morphogenesis to form capillary tubes. We have utilized an in vitro Matrigel overlay assay to analyze the role of the cytoskeleton and Rho GTPases during this process. The addition of matrix first induces changes in cell morphology characterized by the formation of dynamic cellular protrusions and the assembly of discrete aggregates or cords of aligned cells resembling primitive capillary-like structures, but without a recognizable lumen. This is followed by cell migration leading to the formation of a complex interconnecting network of capillary tubes with readily identifiable lumens. Inhibition of actin polymerization or actin-myosin contraction inhibits cell migration but has no effect on the initial changes in endothelial cell morphology. However, inhibition of microtubule dynamics prevents both the initial cell shape changes as well as cell migration. We find that the small GTPase Rac is essential for the matrix-induced changes in endothelial cell morphology, whereas p21-activated kinase, an effector of Rac, is required for cell motility. We conclude that Rac integrates signaling through both the actin and microtubule cytoskeletons to promote capillary tube assembly.

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Faculty Opinions recommendation of Microtubule depolymerization rapidly collapses capillary tube networks in vitro and angiogenic vessels in vivo through the small GTPase Rho.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1017821.210488 ◽

2004 ◽

Author(s):

Andrius Kazlauskas

Keyword(s):

Capillary Tube ◽

Small Gtpase ◽

Microtubule Depolymerization

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Gold Nanoparticles Affect Pericyte Biology and Capillary Tube Formation

Pharmaceutics ◽

10.3390/pharmaceutics13050738 ◽

2021 ◽

Vol 13 (5) ◽

pp. 738

Author(s):

Sasikarn Looprasertkul ◽

Amornpun Sereemaspun ◽

Nakarin Kitkumthorn ◽

Kanidta Sooklert ◽

Tewarit Sarachana ◽

...

Keyword(s):

Gold Nanoparticles ◽

Endothelial Cell ◽

Mrna Expression ◽

Capillary Tube ◽

Quantitative Polymerase Chain Reaction ◽

Tube Formation ◽

Endothelial Cell Proliferation ◽

Control Group ◽

Capillary Tubes ◽

Cell Functions

Gold nanoparticles (AuNPs) are used for diagnostic and therapeutic purposes, especially antiangiogenesis, which are accomplished via inhibition of endothelial cell proliferation, migration, and tube formation. However, no research has been performed on the effects of AuNPs in pericytes, which play vital roles in endothelial cell functions and capillary tube formation during physiological and pathological processes. Therefore, the effects of AuNPs on the morphology and functions of pericytes need to be elucidated. This study treated human placental pericytes in monoculture with 20 nm AuNPs at a concentration of 30 ppm. Ki-67 and platelet-derived growth factor receptor-β (PDGFR-β) mRNA expression was measured using real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell migration was assessed by Transwell migration assay. The fine structures of pericytes were observed by transmission electron microscopy. In addition, 30 ppm AuNP-treated pericytes and intact human umbilical vein endothelial cells were cocultured on Matrigel to form three-dimensional (3D) capillary tubes. The results demonstrated that AuNPs significantly inhibited proliferation, reduced PDGFR-β mRNA expression, and decreased migration in pericytes. Ultrastructural analysis of pericytes revealed AuNPs in late endosomes, autolysosomes, and mitochondria. Remarkably, many mitochondria were swollen or damaged. Additionally, capillary tube formation was reduced. We found that numerous pericytes on 3D capillary tubes were round and did not extend their processes along the tubes, which resulted in more incomplete tube formation in the treatment group compared with the control group. In summary, AuNPs can affect pericyte proliferation, PDGFR-β mRNA expression, migration, morphology, and capillary tube formation. The findings highlight the possible application of AuNPs in pericyte-targeted therapy for antiangiogenesis.

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Ras-mediated activation of the TORC2–PKB pathway is critical for chemotaxis

The Journal of Cell Biology ◽

10.1083/jcb.201001129 ◽

2010 ◽

Vol 190 (2) ◽

pp. 233-245 ◽

Cited By ~ 78

Author(s):

Huaqing Cai ◽

Satarupa Das ◽

Yoichiro Kamimura ◽

Yu Long ◽

Carole A. Parent ◽

...

Keyword(s):

Cell Migration ◽

Actin Polymerization ◽

Time Course ◽

Specific Inhibitor ◽

Ras Proteins ◽

Extracellular Signaling ◽

Upstream Regulator ◽

Pkb Phosphorylation ◽

G Protein Coupled

In chemotactic cells, G protein–coupled receptors activate Ras proteins, but it is unclear how Ras-associated pathways link extracellular signaling to cell migration. We show that, in Dictyostelium discoideum, activated forms of RasC prolong the time course of TORC2 (target of rapamycin [Tor] complex 2)-mediated activation of a myristoylated protein kinase B (PKB; PKBR1) and the phosphorylation of PKB substrates, independently of phosphatidylinositol-(3,4,5)-trisphosphate. Paralleling these changes, the kinetics of chemoattractant-induced adenylyl cyclase activation and actin polymerization are extended, pseudopodial activity is increased and mislocalized, and chemotaxis is impaired. The effects of activated RasC are suppressed by deletion of the TORC2 subunit PiaA. In vitro RasCQ62L-dependent PKB phosphorylation can be rapidly initiated by the addition of a PiaA-associated immunocomplex to membranes of TORC2-deficient cells and blocked by TOR-specific inhibitor PP242. Furthermore, TORC2 binds specifically to the activated form of RasC. These results demonstrate that RasC is an upstream regulator of TORC2 and that the TORC2–PKB signaling mediates effects of activated Ras proteins on the cytoskeleton and cell migration.

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Structure and Function of a Vimentin-associated Matrix Adhesion in Endothelial Cells

Molecular Biology of the Cell ◽

10.1091/mbc.12.1.85 ◽

2001 ◽

Vol 12 (1) ◽

pp. 85-100 ◽

Cited By ~ 111

Author(s):

Meredith Gonzales ◽

Babette Weksler ◽

Daisuke Tsuruta ◽

Robert D. Goldman ◽

Kristine J. Yoon ◽

...

Keyword(s):

Endothelial Cells ◽

Growth Factors ◽

Endothelial Cell ◽

Branching Morphogenesis ◽

Basement Membranes ◽

Αvβ3 Integrin ◽

Dependent Manner ◽

Matrix Adhesion ◽

Focal Contacts

The α4 laminin subunit is a component of endothelial cell basement membranes. An antibody (2A3) against the α4 laminin G domain stains focal contact-like structures in transformed and primary microvascular endothelial cells (TrHBMECs and HMVECs, respectively), provided the latter cells are activated with growth factors. The 2A3 antibody staining colocalizes with that generated by αv and β3 integrin antibodies and, consistent with this localization, TrHBMECs and HMVECs adhere to the α4 laminin subunit G domain in an αvβ3-integrin–dependent manner. The αvβ3 integrin/2A3 antibody positively stained focal contacts are recognized by vinculin antibodies as well as by antibodies against plectin. Unusually, vimentin intermediate filaments, in addition to microfilament bundles, interact with many of the αvβ3 integrin-positive focal contacts. We have investigated the function of α4-laminin and αvβ3-integrin, which are at the core of these focal contacts, in cultured endothelial cells. Antibodies against these proteins inhibit branching morphogenesis of TrHBMECs and HMVECs in vitro, as well as their ability to repopulate in vitro wounds. Thus, we have characterized an endothelial cell matrix adhesion, which shows complex cytoskeletal interactions and whose assembly is regulated by growth factors. Our data indicate that this adhesion structure may play a role in angiogenesis.

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A comparative assessment of e-cigarette aerosols and cigarette smoke on in vitro endothelial cell migration

Toxicology Letters ◽

10.1016/j.toxlet.2017.07.651 ◽

2017 ◽

Vol 280 ◽

pp. S235-S236

Author(s):

Mark Taylor ◽

Tomasz Jaunky ◽

Katherine Hewitt ◽

Frazer Lowe ◽

Ian Fearon ◽

...

Keyword(s):

Cell Migration ◽

Endothelial Cell ◽

Cigarette Smoke ◽

Comparative Assessment ◽

Endothelial Cell Migration

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Vascular endothelial growth factor (VEGF) in cartilage neovascularization and chondrocyte differentiation: auto-paracrine role during endochondral bone formation

Journal of Cell Science ◽

10.1242/jcs.113.1.59 ◽

2000 ◽

Vol 113 (1) ◽

pp. 59-69 ◽

Cited By ~ 8

Author(s):

M.F. Carlevaro ◽

S. Cermelli ◽

R. Cancedda ◽

F. Descalzi Cancedda

Keyword(s):

Endothelial Cells ◽

Cell Migration ◽

Endothelial Cell ◽

Hypertrophic Chondrocytes ◽

Endothelial Cell Migration ◽

Vegf Receptor ◽

Hypertrophic Cartilage ◽

Endothelial Growth Factor

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induces endothelial cell migration and proliferation in culture and is strongly angiogenic in vivo. VEGF synthesis has been shown to occur in both normal and transformed cells. The receptors for the factor have been shown to be localized mainly in endothelial cells, however, the presence of VEGF synthesis and the VEGF receptor in cells other than endothelial cells has been demonstrated. Neoangiogenesis in cartilage growth plate plays a fundamental role in endochondral ossification. We have shown that, in an avian in vitro system for chondrocyte differentiation, VEGF was produced and localized in cell clusters totally resembling in vivo cartilage. The factor was synthesized by hypertrophic chondrocytes and was released into their conditioned medium, which is highly chemotactic for endothelial cells. Antibodies against VEGF inhibited endothelial cell migration induced by chondrocyte conditioned media. Similarly, endothelial cell migration was inhibited also by antibodies directed against the VEGF receptor 2/Flk1 (VEGFR2). In avian and mammalian embryo long bones, immediately before vascular invasion, VEGF was distinctly localized in growth plate hypertrophic chondrocytes. In contrast, VEGF was not observed in quiescent and proliferating chondrocytes earlier in development. VEGF receptor 2 colocalized with the factor both in hypertrophic cartilage in vivo and hypertrophic cartilage engineered in vitro, suggesting an autocrine loop in chondrocytes at the time of their maturation to hypertrophic cells and of cartilage erosion. Regardless of cell exposure to exogenous VEGF, VEGFR-2 phosphorylation was recognized in cultured hypertrophic chondrocytes, supporting the idea of an autocrine functional activation of signal transduction in this non-endothelial cell type as a consequence of the endogenous VEGF production. In summary we propose that VEGF is actively responsible for hypertrophic cartilage neovascularization through a paracrine release by chondrocytes, with invading endothelial cells as a target. Furthermore, VEGF receptor localization and signal transduction in chondrocytes strongly support the hypothesis of a VEGF autocrine activity also in morphogenesis and differentiation of a mesoderm derived cell.

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DARC Regulates Angiogenesis by Mediating Vascular Endothelial Cell Migration

10.21203/rs.3.rs-115340/v1 ◽

2020 ◽

Author(s):

Xiaolin Wang ◽

Yongqian Bian ◽

Yuejun Li ◽

Jing Li ◽

Congying Zhao ◽

...

Keyword(s):

Cell Migration ◽

Endothelial Cell ◽

Vascular Endothelial Cell ◽

Tube Formation ◽

Endothelial Cell Migration ◽

Immunofluorescent Staining ◽

Control Group ◽

Rhoa Activation ◽

And Control

Abstract Background: DARC (The Duffy antigen receptor for chemokines) is a kind of glycosylated membrane protein that binds to members of the CXC chemokine family associated with angiogenesis and has recently been reported to be implicated in diverse normal physiologic processes. This study aimed to investigate the involvement of DARC in angiogenesis, which is known to generate new capillary blood vessels from preexisting ones. Methods: HDMECs (Human dermal microvascular endothelial cells) were divided into two groups (DARC overexpression group, and control group). We used Brdu staining to detect cell proliferation, and wound healing assay to detect cell migration. Then tube formation assay were observed. Also, western blot and immunofluorescent staining were used to estimate the relationship between DARC and RhoA (Ras homolog gene family, member A). Results: HDMECs proliferation, migration, and tube formation were inhibited significantly when DARC was overexpressed intracellular. DARC impaired microfilament dynamics and intercellular connection in migrating cells, and RhoA activation underlay the effect of DARC on endothelial cell. Furthermore, DARC inhibited the formation of new capillaries in vitro. Conclusion: Our ﬁndings revealed the role of DARC in the angiogenic process and provided a novel mechanism for RhoA activation during endothelial cell migration and angiogenesis.

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PLATELET DEPOSITION ON ARTIFICIAL SURFACES: EFFECT OF PROTEIN PRECOATING, ENDOTHELIAL CELL COVERAGE AND SHEAR RATE

10.1055/s-0038-1642917 ◽

1987 ◽

Author(s):

A Poot ◽

A Dekker ◽

T Beugeling ◽

A Bantjes ◽

W G Van Aken

Keyword(s):

Endothelial Cell ◽

Shear Rate ◽

Capillary Tube ◽

Capillary Perfusion ◽

Perfusate Flow ◽

Platelet Deposition ◽

Artificial Surface ◽

Platelet Spreading ◽

Von Willebrand

In the present study the in vitro capillary perfusion system according to Cazenave was used. This system consists of a capillary tube connected to a syringe containg the perfusate which consisted of washed human platalet (111 In-labeled or native) and washed red cells in Ca2+ /Mg2+ -containing Tyrode-albumin buffer. Perfusate flow is controlled by a syringe pump. Polyethylene tubes (PE, 0.75 mm ID, 25 cm long) were precoated with purified human von Willebrand factor (vWF), fibrinogen (Fb), fibronectin (Fn), immunoglobulin G (IgG), albumin (HSA) or plasma. Compared with uncoated PE, platelet deposition increased after precoating with vWF, Fb or Fn, and decreased by pre adsorbed IgG, HSA or plasma. Platelet deposition was positively correlated with shear rate only on surfaces precoated with vWF, Fb or Fn. Scanning electron microscopy showed platelet aggregates on IgG—coated PE, whereas on all other surfaces single adherent platelets were observed. Complete platelet spreading was only observed after precoating with Fn. In contrast with PE coated with vWF, Fb or Fn, platelet adhesion on uncoated PE did not increase further after 5 minutes of perfusion probably due to passivation of the surface by albumin present in the perfusate. This could be overcome by addition of Fb to the perfusate. Perfusates prepared in this way were used for studying the effect of human endothelial cell (HEC) coverage on platelet deposition. HEC were seeded at different densities in PE tubes precoated with partially purified Fn. Platelet deposition decreased with 2increasing HEC coverage, and was negligible at 60,000 HEC/cm . Our results indicate the applicability of this perfusion model for the in vitro testing of artificial surface thrombogenicity.

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