Endothelial cell invasion is controlled by dactylopodia

Sprouting angiogenesis is fundamental for development and contributes to cancer, diabetic retinopathy, and cardiovascular diseases. Sprouting angiogenesis depends on the invasive properties of endothelial tip cells. However, there is very limited knowledge on how tip cells invade into tissues. Here, we show that endothelial tip cells use dactylopodia as the main cellular protrusion for invasion into nonvascular extracellular matrix. We show that dactylopodia and filopodia protrusions are balanced by myosin IIA (NMIIA) and actin-related protein 2/3 (Arp2/3) activity. Endothelial cell-autonomous ablation of NMIIA promotes excessive dactylopodia formation in detriment of filopodia. Conversely, endothelial cell-autonomous ablation of Arp2/3 prevents dactylopodia development and leads to excessive filopodia formation. We further show that NMIIA inhibits Rac1-dependent activation of Arp2/3 by regulating the maturation state of focal adhesions. Our discoveries establish a comprehensive model of how endothelial tip cells regulate its protrusive activity and will pave the way toward strategies to block invasive tip cells during sprouting angiogenesis.

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Endothelial cell invasiveness is controlled by myosin IIA-dependent inhibition of Arp2/3 activity

10.1101/2020.09.08.287466 ◽

2020 ◽

Author(s):

Ana M. Figueiredo ◽

Pedro Barbacena ◽

Ana Russo ◽

Silvia Vaccaro ◽

Daniela Ramalho ◽

...

Keyword(s):

Endothelial Cell ◽

Focal Adhesions ◽

Sprouting Angiogenesis ◽

Dependent Inhibition ◽

Myosin Iia ◽

Maturation State ◽

Cell Invasiveness ◽

Cellular Protrusion ◽

Tip Cells ◽

New Strategies

AbstractSprouting angiogenesis is fundamental for development and contributes to multiple diseases, including cancer, diabetic retinopathy and cardiovascular diseases. Sprouting angiogenesis depends on the invasive properties of endothelial tip cells. However, there is very limited knowledge on the mechanisms that endothelial tip cells use to invade into tissues. Here, we prove that endothelial tip cells use long lamellipodia projections (LLPs) as the main cellular protrusion for invasion into non-vascular extracellular matrix. We show that LLPs and filopodia protrusions are balanced by myosin-IIA (MIIA) and actin-related protein 2/3 (Arp2/3) activity. Endothelial cell-autonomous ablation of MIIA promotes excessive LLPs formation in detriment of filopodia. Conversely, endothelial cell-autonomous ablation of Arp2/3 prevents LLPs development and leads to excessive filopodia formation. We further show that MIIA inhibits Rac1-dependent activation of Arp2/3, by regulating the maturation state of focal adhesions. Our discoveries establish the first comprehensive model of how endothelial tip cells regulate its protrusive activity and will pave the way towards new strategies to block invasive tip cells during sprouting angiogenesis.

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Smooth muscle cell rigidity and extracellular matrix organization influence endothelial cell spreading and adhesion formation in coculture

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00618.2007 ◽

2007 ◽

Vol 293 (3) ◽

pp. H1978-H1986 ◽

Cited By ~ 22

Author(s):

Charles S. Wallace ◽

Sophie A. Strike ◽

George A. Truskey

Keyword(s):

Extracellular Matrix ◽

Smooth Muscle ◽

Endothelial Cell ◽

Focal Adhesion ◽

Adhesion Formation ◽

Focal Adhesions ◽

Cell Spreading ◽

Tissue Engineered Blood Vessels ◽

Focal Adhesion Formation ◽

Fibrillar Adhesion

Efforts to develop functional tissue-engineered blood vessels have focused on improving the strength and mechanical properties of the vessel wall, while the functional status of the endothelium within these vessels has received less attention. Endothelial cell (EC) function is influenced by interactions between its basal surface and the underlying extracellular matrix. In this study, we utilized a coculture model of a tissue-engineered blood vessel to evaluate EC attachment, spreading, and adhesion formation to the extracellular matrix on the surface of quiescent smooth muscle cells (SMCs). ECs attached to and spread on SMCs primarily through the α5β1-integrin complex, whereas ECs used either α5β1- or αvβ3-integrin to spread on fibronectin (FN) adsorbed to plastic. ECs in coculture lacked focal adhesions, but EC α5β1-integrin bound to fibrillar FN on the SMC surface, promoting rapid fibrillar adhesion formation. As assessed by both Western blot analysis and quantitative real-time RT-PCR, coculture suppressed the expression of focal adhesion proteins and mRNA, whereas tensin protein and mRNA expression were elevated. When attached to polyacrylamide gels with similar elastic moduli as SMCs, focal adhesion formation and the rate of cell spreading increased relative to ECs in coculture. Thus, the elastic properties are only one factor contributing to EC spreading and focal adhesion formation in coculture. The results suggest that the softness of the SMCs and the fibrillar organization of FN inhibit focal adhesions and reduce cell spreading while promoting fibrillar adhesion formation. These changes in the type of adhesions may alter EC signaling pathways in tissue-engineered blood vessels.

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The Transcription Factor Complex LMO2/TAL1 Regulates Branching and Endothelial Cell Migration in Sprouting Angiogenesis

10.21203/rs.3.rs-384580/v1 ◽

2021 ◽

Author(s):

Yoshihiro Yamada ◽

Yi Zhong ◽

Shiho Miki ◽

Akiko Taura ◽

Terence Rabbitts

Keyword(s):

Endothelial Cells ◽

Transcription Factor ◽

Endothelial Cell ◽

Endothelial Cell Migration ◽

Angiogenic Switch ◽

Sprouting Angiogenesis ◽

Relative Paucity ◽

Angiopoietin 2 ◽

Tip Cells ◽

Transcription Factor Complex

Abstract The transcription factor complex, consisting of LMO2, TAL1/LYL1, and GATA2, plays an important role in capillary sprouting by regulating VEGFR2, DLL4, and angiopoietin 2 in tip cells. Overexpression of the basic helix-loophelix transcription factor LYL1 in transgenic mice results in shortened tails. This phenotype is associated with vessel hyperbranching and a relative paucity of straight vessels due to DLL4 downregulation in tip cells by forming aberrant complex consisting of LMO2 and LYL1. Knockdown of LMO2 or TAL1 inhibits capillary sprouting in spheroid-based angiogenesis assays, which is associated with decreased angiopoietin 2 secretion. In the same assay using mixed TAL1- and LYL1-expressing endothelial cells, TAL1 was found to be primarily located in tip cells, while LYL1-expressing cells tended to occupy the stalk position in sprouts by upregulating VEGFR1 than TAL1. Thus, the interaction between LMO2 and TAL1 in tip cells plays a key role in angiogenic switch of sprouting angiogenesis.

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Notch1 controls macrophage recruitment and Notch signaling is activated at sites of endothelial cell anastomosis during retinal angiogenesis in mice

Blood ◽

10.1182/blood-2010-12-327015 ◽

2011 ◽

Vol 118 (12) ◽

pp. 3436-3439 ◽

Cited By ~ 84

Author(s):

Hasina H. Outtz ◽

Ian W. Tattersall ◽

Natalie M. Kofler ◽

Nicole Steinbach ◽

Jan Kitajewski

Keyword(s):

Endothelial Cell ◽

Notch Signaling ◽

Leading Edge ◽

Vascular Differentiation ◽

Vascular Integrity ◽

Sprouting Angiogenesis ◽

Macrophage Recruitment ◽

Reporter Mice ◽

Retinal Angiogenesis ◽

Tip Cells

Abstract Notch is a critical regulator of angiogenesis, vascular differentiation, and vascular integrity. We investigated whether Notch signaling affects macrophage function during retinal angiogenesis in mice. Retinal macrophage recruitment and localization in mice with myeloid-specific loss of Notch1 was altered, as these macrophages failed to localize at the leading edge of the vascular plexus and at vascular branchpoints. Furthermore, these retinas were characterized by elongated endothelial cell sprouts that failed to anastomose with neighboring sprouts. Using Notch reporter mice, we demonstrate that retinal macrophages localize between Dll4-positive tip cells and at vascular branchpoints, and that these macrophages had activated Notch signaling. Taken together, these data demonstrate that Notch signaling in macrophages is important for their localization and interaction with endothelial cells during sprouting angiogenesis.

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Septin2 mediates podosome maturation and endothelial cell invasion associated with angiogenesis

The Journal of Cell Biology ◽

10.1083/jcb.201903023 ◽

2019 ◽

Vol 219 (2) ◽

Author(s):

Kerrie B. Collins ◽

Hojin Kang ◽

Jacob Matsche ◽

Jennifer E. Klomp ◽

Jalees Rehman ◽

...

Keyword(s):

Extracellular Matrix ◽

Plasma Membrane ◽

Endothelial Cell ◽

Cell Invasion ◽

Scaffolding Protein ◽

Matrix Remodeling ◽

Close Proximity ◽

Basolateral Plasma Membrane ◽

Binding Residues ◽

And Function

Podosomes are compartmentalized actin-rich adhesions, defined by their ability to locally secrete proteases and remodel extracellular matrix. Matrix remodeling by endothelial podosomes facilitates invasion and thereby vessel formation. However, the mechanisms underlying endothelial podosome formation and function remain unclear. Here, we demonstrate that Septin2, Septin6, and Septin7 are required for maturation of nascent endothelial podosomes into matrix-degrading organelles. We show that podosome development occurs through initial mobilization of the scaffolding protein Tks5 and F-actin accumulation, followed by later recruitment of Septin2. Septin2 localizes around the perimeter of podosomes in close proximity to the basolateral plasma membrane, and phosphoinositide-binding residues of Septin2 are required for podosome function. Combined, our results suggest that the septin cytoskeleton forms a diffusive barrier around nascent podosomes to promote their maturation. Finally, we show that Septin2-mediated regulation of podosomes is critical for endothelial cell invasion associated with angiogenesis. Therefore, targeting of Septin2-mediated podosome formation is a potentially attractive anti-angiogenesis strategy.

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Prostate-Specific Membrane Antigen Regulates Angiogenesis by Modulating Integrin Signal Transduction

Molecular and Cellular Biology ◽

10.1128/mcb.00084-06 ◽

2006 ◽

Vol 26 (14) ◽

pp. 5310-5324 ◽

Cited By ~ 106

Author(s):

Rebecca E. Conway ◽

Nenad Petrovic ◽

Zhong Li ◽

Warren Heston ◽

Dianqing Wu ◽

...

Keyword(s):

Extracellular Matrix ◽

Endothelial Cell ◽

Cell Invasion ◽

Principal Component ◽

Integrin Signaling ◽

Prostate Specific Membrane Antigen ◽

Filamin A ◽

Regulatory Loop ◽

Specific Membrane

ABSTRACT The transmembrane peptidase prostate-specific membrane antigen (PSMA) is universally upregulated in the vasculature of solid tumors, but its functional role in tumor angiogenesis has not been investigated. Here we show that angiogenesis is severely impaired in PSMA-null animals and that this angiogenic defect occurs at the level of endothelial cell invasion through the extracellular matrix barrier. Because proteolytic degradation of the extracellular matrix is a critical component of endothelial invasion in angiogenesis, it is logical to assume that PSMA participates in matrix degradation. However, we demonstrate a novel and more complex role for PSMA in angiogenesis, where it is a principal component of a regulatory loop that is tightly modulating laminin-specific integrin signaling and GTPase-dependent, p21-activated kinase 1 (PAK-1) activity. We show that PSMA inhibition, knockdown, or deficiency decreases endothelial cell invasion in vitro via integrin and PAK, thus abrogating angiogenesis. Interestingly, the neutralization of β1 or the inactivation of PAK increases PSMA activity, suggesting that they negatively regulate PSMA. This negative regulation is mediated by the cytoskeleton as the disruption of interactions between the PSMA cytoplasmic tail and the anchor protein filamin A decreases PSMA activity, integrin function, and PAK activation. Finally, the inhibition of PAK activation enhances the PSMA/filamin A interaction and, thus, boosts PSMA activity. These data imply that PSMA participates in an autoregulatory loop, wherein active PSMA facilitates integrin signaling and PAK activation, leading to both productive invasion and downregulation of integrin β1 signaling via reduced PSMA activity. Therefore, we have identified a novel role for PSMA as a true molecular interface, integrating both extracellular and intracellular signals during angiogenesis.

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Endothelial cell regulation of matrix metalloproteinases

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y04-120 ◽

2005 ◽

Vol 83 (1) ◽

pp. 1-7 ◽

Cited By ~ 50

Author(s):

Tara L Haas

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Endothelial Cell ◽

Matrix Metalloproteinases ◽

Basement Membrane ◽

Matrix Proteins ◽

Regulation Of Transcription ◽

Cell Type ◽

Sprouting Angiogenesis

The process of sprouting angiogenesis requires that the endothelial cells degrade the basement membrane matrix and migrate into the interstitial matrix. Matrix metalloproteinases are enzymes capable of cleaving numerous extracellular matrix proteins. Increased production and activity of matrix metalloproteinases in any cell type is associated with a more migratory and invasive phenotype. This paper describes results of recent in-vitro studies of the regulation of transcription and activation of MMP-2 and MT1-MMP in endothelial cells, as well as studies that examined roles of matrix metalloproteinases in activity-induced angiogenesis.Key words: proteolysis, extracellular matrix, angiogenesis, mechanotransduction.

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eNOS controls angiogenic sprouting and retinal neovascularization through the regulation of endothelial cell polarity

Cellular and Molecular Life Sciences ◽

10.1007/s00018-021-04042-y ◽

2021 ◽

Author(s):

Tracy L. Smith ◽

Malika Oubaha ◽

Gael Cagnone ◽

Cécile Boscher ◽

Jin Sung Kim ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cell Polarity ◽

Cell Polarization ◽

No Synthase ◽

Sprouting Angiogenesis ◽

Oxygen Induced Retinopathy ◽

Tip Cells ◽

Deficient Mice ◽

Vascular Branching

AbstractThe roles of nitric oxide (NO) and endothelial NO synthase (eNOS) in the regulation of angiogenesis are well documented. However, the involvement of eNOS in the sprouting of endothelial tip-cells at the vascular front during sprouting angiogenesis remains poorly defined. In this study, we show that downregulation of eNOS markedly inhibits VEGF-stimulated migration of endothelial cells but increases their polarization, as evidenced by the reorientation of the Golgi in migrating monolayers and by the fewer filopodia on tip cells at ends of sprouts in endothelial cell spheroids. The effect of eNOS inhibition on EC polarization was prevented in Par3-depleted cells. Importantly, downregulation of eNOS increased the expression of polarity genes, such as PARD3B, PARD6A, PARD6B, PKCΖ, TJP3, and CRB1 in endothelial cells. In retinas of eNOS knockout mice, vascular development is retarded with decreased vessel density and vascular branching. Furthermore, tip cells at the extremities of the vascular front have a marked reduction in the number of filopodia per cell and are more oriented. In a model of oxygen-induced retinopathy (OIR), eNOS deficient mice are protected during the initial vaso-obliterative phase, have reduced pathological neovascularization, and retinal endothelial tip cells have fewer filopodia. Single-cell RNA sequencing of endothelial cells from OIR retinas revealed enrichment of genes related to cell polarity in the endothelial tip-cell subtype of eNOS deficient mice. These results indicate that inhibition of eNOS alters the polarity program of endothelial cells, which increases cell polarization, regulates sprouting angiogenesis and normalizes pathological neovascularization during retinopathy.

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Aged Breast Extracellular Matrix Drives Mammary Epithelial Cells to an Invasive and Cancer-Like Phenotype

10.1101/2020.09.30.320960 ◽

2020 ◽

Cited By ~ 1

Author(s):

Gokhan Bahcecioglu ◽

Xiaoshan Yue ◽

Erin Howe ◽

Ian Guldner ◽

M. Sharon Stack ◽

...

Keyword(s):

Extracellular Matrix ◽

Epithelial Cells ◽

Cell Motility ◽

Cell Invasion ◽

Protein Production ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Membrane Localization ◽

Related Protein ◽

E Cadherin

AbstractAge is a major risk factor for cancer. While the importance of age related genetic alterations in cells on cancer progression is well documented, the effect of aging extracellular matrix (ECM) has been overlooked. Here, we show for the first time that the aging breast ECM is sufficient to drive normal mammary epithelial cells (KTB21) to a more invasive and cancer-like phenotype, while promoting motility and invasiveness in MDA-MB-231 cells. E-cadherin membrane localization was lost in KTB21 cells cultured on the decellularized breast matrix from aged mice. Cell motility, cell invasion, and inflammatory cytokine and cancer-related protein production were increased significantly on the aged matrix, and many genes related to invasion were upregulated. Strikingly, we showed using single cell RNA sequencing that the aged matrix led to enrichment of a subpopulation of KTB21 cells that highly expressed epithelial-mesenchymal transition (EMT) and invasion-related genes. Lysyl oxidase (LOX) knockdown reverted the aged matrix-induced changes to the young levels; LOX siRNA treatment prevented the loss of E-cadherin membrane localization, and reduced cell motility, cell invasion, and cytokine and cancer-related protein production. Finally, we showed that the biophysical, mechanical and biochemical properties of the breast ECM were altered dramatically upon aging. Analyzing these factors and studying the differential response of the epithelial cells to young and aged ECMs could lead to identification of new targets for cancer treatment and could pave the way for the discovery of new therapeutic options.

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Naturally Produced Extracellular Matrix Is an Excellent Substrate for Canine Endothelial Cell Proliferation and Resistance to Shear Stress on PTFE Vascular Grafts

Thrombosis and Haemostasis ◽

10.1055/s-0038-1665417 ◽

1997 ◽

Vol 78 (05) ◽

pp. 1392-1398 ◽

Cited By ~ 16

Author(s):

A Schneider ◽

M Chandra ◽

G Lazarovici ◽

I Vlodavsky ◽

G Merin ◽

...

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Shear Stress ◽

Endothelial Cell ◽

Cell Attachment ◽

Thrombus Formation ◽

Endothelial Cell Proliferation ◽

Ptfe Graft ◽

Vascular Prostheses ◽

Endothelial Cell Attachment

SummaryPurpose: Successful development of a vascular prosthesis lined with endothelial cells (EC) may depend on the ability of the attached cells to resist shear forces after implantation. The present study was designed to investigate EC detachment from extracellular matrix (ECM) precoated vascular prostheses, caused by shear stress in vitro and to test the performance of these grafts in vivo. Methods: Bovine aortic endothelial cells were seeded inside untreated polytetrafluoro-ethylene (PTFE) vascular graft (10 X 0.6 cm), PTFE graft precoated with fibronectin (FN), or PTFE precoated with FN and a naturally produced ECM (106 cells/graft). Sixteen hours after seeding the medium was replaced and unattached cells counted. The strength of endothelial cell attachment was evaluated by subjecting the grafts to a physiologic shear stress of 15 dynes/cm2 for 1 h. The detached cells were collected and quantitated. PTFE or EC preseeded ECM coated grafts were implanted in the common carotid arteries of dogs. Results: While little or no differences were found in the extent of endothelial cell attachment to the various grafts (79%, 87% and 94% of the cells attached to PTFE, FN precoated PTFE, or FN+ECM precoated PTFE, respectively), the number of cells retained after a shear stress was significanly increased on ECM coated PTFE (20%, 54% and 85% on PTFE, FN coated PTFE, and FN+ECM coated PTFE, respectively, p <0.01). Implantation experiments in dogs revealed a significant increase in EC coverage and a reduced incidence of thrombus formation on ECM coated grafts that were seeded with autologous saphenous vein endothelial cells prior to implantation. Conclusion: ECM coating significantly increased the strength of endothelial cell attachment to vascular prostheses subjected to shear stress. The presence of adhesive macromolecules and potent endothelial cell growth promoting factors may render the ECM a promising substrate for vascular prostheses.

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