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1.
J Cell Sci ; 137(13)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38881365

RESUMO

Endothelial cells lining the blood vessel wall communicate intricately with the surrounding extracellular matrix, translating mechanical cues into biochemical signals. Moreover, vessels require the capability to enzymatically degrade the matrix surrounding them, to facilitate vascular expansion. c-Src plays a key role in blood vessel growth, with its loss in the endothelium reducing vessel sprouting and focal adhesion signalling. Here, we show that constitutive activation of c-Src in endothelial cells results in rapid vascular expansion, operating independently of growth factor stimulation or fluid shear stress forces. This is driven by an increase in focal adhesion signalling and size, with enhancement of localised secretion of matrix metalloproteinases responsible for extracellular matrix remodelling. Inhibition of matrix metalloproteinase activity results in a robust rescue of the vascular expansion elicited by heightened c-Src activity. This supports the premise that moderating focal adhesion-related events and matrix degradation can counteract abnormal vascular expansion, with implications for pathologies driven by unusual vascular morphologies.


Assuntos
Matriz Extracelular , Adesões Focais , Quinases da Família src , Adesões Focais/metabolismo , Matriz Extracelular/metabolismo , Humanos , Quinases da Família src/metabolismo , Quinases da Família src/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Animais , Proteína Tirosina Quinase CSK/metabolismo , Transdução de Sinais , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Metaloproteinases da Matriz/metabolismo
2.
STAR Protoc ; 4(4): 102626, 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-37792537

RESUMO

Adherens junctions (AJs) are multi-protein adhesion structures that couple contractile actomyosin networks of epithelial cells within a tissue. Here, we present an epithelial cell spreading assay on E-cadherin-coated glass or polydimethylsiloxane (PDMS) substrates for detailed microscopy-based analysis of cadherin adhesions. We describe steps for preparation of glass coverslips and PDMS gels, E-cadherin coating, and epithelial cell spreading. Epithelial cells can be seeded on E-cadherin-coated surfaces, thereby mimicking AJ formation in X-Y dimension, making it suitable for microscopy analysis. For complete details on the use and execution of this protocol, please refer to Noordstra et al. (2023).1.


Assuntos
Caderinas , Microscopia , Caderinas/química , Caderinas/metabolismo , Células Epiteliais , Junções Aderentes/metabolismo , Dimetilpolisiloxanos
3.
Dev Cell ; 58(18): 1748-1763.e6, 2023 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-37480844

RESUMO

Adherens junctions (AJs) allow cell contact to inhibit epithelial migration yet also permit epithelia to move as coherent sheets. How, then, do cells identify which contacts will inhibit locomotion? Here, we show that in human epithelial cells this arises from the orientation of cortical flows at AJs. When the leader cells from different migrating sheets make head-on contact with one another, they assemble AJs that couple together oppositely directed cortical flows. This applies a tensile signal to the actin-binding domain (ABD) of α-catenin, which provides a clutch to promote lateral adhesion growth and inhibit the lamellipodial activity necessary for migration. In contrast, AJs found between leader cells in the same migrating sheet have cortical flows aligned in the same direction, and no such mechanical inhibition takes place. Therefore, α-catenin mechanosensitivity in the clutch between E-cadherin and cortical F-actin allows cells to interpret the direction of motion via cortical flows and signal for contact to inhibit locomotion.


Assuntos
Actinas , Locomoção , Humanos , alfa Catenina , Caderinas , Células Epiteliais
4.
Nat Cardiovasc Res ; 1(12): 1156-1173, 2022 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-37936984

RESUMO

Vascular endothelial (VE)-cadherin in endothelial adherens junctions is an essential component of the vascular barrier, critical for tissue homeostasis and implicated in diseases such as cancer and retinopathies. Inhibitors of Src cytoplasmic tyrosine kinase have been applied to suppress VE-cadherin tyrosine phosphorylation and prevent excessive leakage, edema and high interstitial pressure. Here we show that the Src-related Yes tyrosine kinase, rather than Src, is localized at endothelial cell (EC) junctions where it becomes activated in a flow-dependent manner. EC-specific Yes1 deletion suppresses VE-cadherin phosphorylation and arrests VE-cadherin at EC junctions. This is accompanied by loss of EC collective migration and exaggerated agonist-induced macromolecular leakage. Overexpression of Yes1 causes ectopic VE-cadherin phosphorylation, while vascular leakage is unaffected. In contrast, in EC-specific Src-deficiency, VE-cadherin internalization is maintained, and leakage is suppressed. In conclusion, Yes-mediated phosphorylation regulates constitutive VE-cadherin turnover, thereby maintaining endothelial junction plasticity and vascular integrity.

5.
Clin Transl Immunology ; 10(10): e1350, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34721846

RESUMO

OBJECTIVES: Thrombotic and microvascular complications are frequently seen in deceased COVID-19 patients. However, whether this is caused by direct viral infection of the endothelium or inflammation-induced endothelial activation remains highly contentious. METHODS: Here, we use patient autopsy samples, primary human endothelial cells and an in vitro model of the pulmonary epithelial-endothelial cell barrier. RESULTS: We show that primary human endothelial cells express very low levels of the SARS-CoV-2 receptor ACE2 and the protease TMPRSS2, which blocks their capacity for productive viral infection, and limits their capacity to produce infectious virus. Accordingly, endothelial cells can only be infected when they overexpress ACE2, or are exposed to very high concentrations of SARS-CoV-2. We also show that SARS-CoV-2 does not infect endothelial cells in 3D vessels under flow conditions. We further demonstrate that in a co-culture model endothelial cells are not infected with SARS-CoV-2. Endothelial cells do however sense and respond to infection in the adjacent epithelial cells, increasing ICAM-1 expression and releasing pro-inflammatory cytokines. CONCLUSIONS: Taken together, these data suggest that in vivo, endothelial cells are unlikely to be infected with SARS-CoV-2 and that infection may only occur if the adjacent pulmonary epithelium is denuded (basolateral infection) or a high viral load is present in the blood (apical infection). In such a scenario, whilst SARS-CoV-2 infection of the endothelium can occur, it does not contribute to viral amplification. However, endothelial cells may still play a key role in SARS-CoV-2 pathogenesis by sensing adjacent infection and mounting a pro-inflammatory response to SARS-CoV-2.

6.
Elife ; 102021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34431475

RESUMO

Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, alhough it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.


Assuntos
Regulação da Expressão Gênica/fisiologia , Junções Intercelulares/fisiologia , Neutrófilos/fisiologia , Animais , Linhagem Celular , Proteínas de Fluorescência Verde , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Músculo Esquelético/fisiologia , Músculo Esquelético/ultraestrutura
7.
Front Immunol ; 12: 667213, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34084168

RESUMO

An inflammatory response requires leukocytes to migrate from the circulation across the vascular lining into the tissue to clear the invading pathogen. Whereas a lot of attention is focused on how leukocytes make their way through the endothelial monolayer, it is less clear how leukocytes migrate underneath the endothelium before they enter the tissue. Upon finalization of the diapedesis step, leukocytes reside in the subendothelial space and encounter endothelial focal adhesions. Using TIRF microscopy, we show that neutrophils navigate around these focal adhesions. Neutrophils recognize focal adhesions as physical obstacles and deform to get around them. Increasing the number of focal adhesions by silencing the small GTPase RhoJ slows down basolateral crawling of neutrophils. However, apical crawling and diapedesis itself are not affected by RhoJ depletion. Increasing the number of focal adhesions drastically by expressing the Rac1 GEF Tiam1 make neutrophils to avoid migrating underneath these Tiam1-expressing endothelial cells. Together, our results show that focal adhesions mark the basolateral migration path of neutrophils.


Assuntos
Células Endoteliais/fisiologia , Adesões Focais/fisiologia , Neutrófilos/fisiologia , Migração Transendotelial e Transepitelial/fisiologia , Linhagem Celular , Humanos , Leucócitos/fisiologia , Cordão Umbilical/patologia
8.
Front Physiol ; 11: 684, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32625119

RESUMO

Blood and lymphatic vessels are lined by endothelial cells which constantly interact with their luminal and abluminal extracellular environments. These interactions confer physical forces on the endothelium, such as shear stress, stretch and stiffness, to mediate biological responses. These physical forces are often altered during disease, driving abnormal endothelial cell behavior and pathology. Therefore, it is critical that we understand the mechanisms by which endothelial cells respond to physical forces. Traditionally, endothelial cells in culture are grown in the absence of flow on stiff substrates such as plastic or glass. These cells are not subjected to the physical forces that endothelial cells endure in vivo, thus the results of these experiments often do not mimic those observed in the body. The field of vascular biology now realize that an intricate analysis of endothelial signaling mechanisms requires complex in vitro systems to mimic in vivo conditions. Here, we will review what is known about the mechanical forces that guide endothelial cell behavior and then discuss the advancements in endothelial cell culture models designed to better mimic the in vivo vascular microenvironment. A wider application of these technologies will provide more biologically relevant information from cultured cells which will be reproducible to conditions found in the body.

9.
Development ; 147(7)2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32108024

RESUMO

Endothelial cell adhesion is implicated in blood vessel sprout formation, yet how adhesion controls angiogenesis, and whether it occurs via rapid remodeling of adherens junctions or focal adhesion assembly, or both, remains poorly understood. Furthermore, how endothelial cell adhesion is controlled in particular tissues and under different conditions remains unexplored. Here, we have identified an unexpected role for spatiotemporal c-Src activity in sprouting angiogenesis in the retina, which is in contrast to the dominant focus on the role of c-Src in the maintenance of vascular integrity. Thus, mice specifically deficient in endothelial c-Src displayed significantly reduced blood vessel sprouting and loss in actin-rich filopodial protrusions at the vascular front of the developing retina. In contrast to what has been observed during vascular leakage, endothelial cell-cell adhesion was unaffected by loss of c-Src. Instead, decreased angiogenic sprouting was due to loss of focal adhesion assembly and cell-matrix adhesion, resulting in loss of sprout stability. These results demonstrate that c-Src signaling at specified endothelial cell membrane compartments (adherens junctions or focal adhesions) control vascular processes in a tissue- and context-dependent manner.


Assuntos
Adesão Celular/genética , Células Endoteliais/fisiologia , Adesões Focais/genética , Genes src/fisiologia , Neovascularização Fisiológica/genética , Retina/embriologia , Animais , Células Cultivadas , Embrião de Mamíferos , Endotélio Vascular/embriologia , Endotélio Vascular/metabolismo , Feminino , Adesões Focais/metabolismo , Adesões Focais/fisiologia , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Knockout , Retina/metabolismo
10.
J Cell Sci ; 133(3)2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-31964713

RESUMO

Endothelial YAP/TAZ (YAP is also known as YAP1, and TAZ as WWTR1) signaling is crucial for sprouting angiogenesis and vascular homeostasis. However, the underlying molecular mechanisms that explain how YAP/TAZ control the vasculature remain unclear. This study reveals that the focal adhesion protein deleted-in-liver-cancer 1 (DLC1) is a direct transcriptional target of the activated YAP/TAZ-TEAD complex. We find that substrate stiffening and VEGF stimuli promote expression of DLC1 in endothelial cells. In turn, DLC1 expression levels are YAP and TAZ dependent, and constitutive activation of YAP is sufficient to drive DLC1 expression. DLC1 is needed to limit F-actin fiber formation, integrin-based focal adhesion lifetime and integrin-mediated traction forces. Depletion of endothelial DLC1 strongly perturbs cell polarization in directed collective migration and inhibits the formation of angiogenic sprouts. Importantly, ectopic expression of DLC1 is sufficient to restore migration and angiogenic sprouting in YAP-depleted cells. Together, these findings point towards a crucial and prominent role for DLC1 in YAP/TAZ-driven endothelial adhesion remodeling and collective migration during angiogenesis.This article has an associated First Person interview with the first author of the paper.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Células Endoteliais , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Endoteliais/metabolismo , Proteínas Ativadoras de GTPase/genética , Humanos , Morfogênese , Neovascularização Patológica , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/genética
11.
Small GTPases ; 11(3): 225-232, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-28960175

RESUMO

Active remodeling of the actin cytoskeleton in endothelial cells is necessary for allowing leukocytes to cross the barrier during the process of transendothelial migration (TEM). Involvement of RhoGTPases to regulate actin organization is inevitable, and we recently reported on the local function of RhoA in limiting vascular leakage during leukocyte TEM. As a follow-up we investigated here the possible involvement of two other closely-related GTPases; RhoB and RhoC, in regulating leukocyte TEM and vascular barrier maintenance. Physiological flow experiments showed no substantial involvement of either endothelial RhoB or RhoC in neutrophil adhesion and transmigration efficiency. Besides neutrophil TEM, we did not observe a role for endothelial RhoB or RhoC in limiting vascular leakage in both inflammatory conditions and during TEM. In conclusion, endothelial RhoB and RhoC are both dispensable for regulating leukocyte diapedesis and for maintaining vascular barrier function under inflammatory conditions and during leukocyte diapedesis.


Assuntos
Células Endoteliais da Veia Umbilical Humana/metabolismo , Leucócitos/metabolismo , Proteína rhoB de Ligação ao GTP/metabolismo , Proteína de Ligação a GTP rhoC/metabolismo , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Leucócitos/citologia , Migração Transendotelial e Transepitelial
12.
Biochem Soc Trans ; 46(6): 1673-1680, 2018 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-30514769

RESUMO

Endothelial cell-cell adhesion within the wall of the vasculature controls a range of physiological processes, such as growth, integrity and barrier function. The adhesive properties of endothelial cells are tightly controlled by a complex cascade of signals transmitted from the surrounding environment or from within the cells themselves, with the dynamic nature of cellular adhesion and the regulating signalling networks now beginning to be appreciated. Here, we summarise the current knowledge of the mechanisms controlling endothelial cell-cell adhesion in the developing and mature blood vasculature.


Assuntos
Adesão Celular/fisiologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Animais , Humanos , Neovascularização Fisiológica/fisiologia , Transdução de Sinais/fisiologia
13.
Cell Rep ; 24(12): 3115-3124, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30231995

RESUMO

Leukocytes follow the well-defined steps of rolling, spreading, and crawling prior to diapedesis through endothelial cells (ECs). We found increased expression of DLC-1 in stiffness-associated diseases like atherosclerosis and pulmonary arterial hypertension. Depletion of DLC-1 in ECs cultured on stiff substrates drastically reduced cell stiffness and mimicked leukocyte transmigration kinetics observed for ECs cultured on soft substrates. Mechanistic studies revealed that DLC-1-depleted ECs or ECs cultured on soft substrates failed to recruit the actin-adaptor proteins filamin B, α-actinin-4, and cortactin to clustered ICAM-1, thereby preventing the ICAM-1 adhesome formation and impairing leukocyte spreading. This was rescued by overexpressing DLC-1, resulting in ICAM-1 adhesome stabilization and leukocyte spreading. Our results reveal an essential role for substrate stiffness-regulated endothelial DLC-1, independent of its GAP domain, in locally stabilizing the ICAM-1 adhesome to promote leukocyte spreading, essential for efficient leukocyte transendothelial migration.


Assuntos
Proteínas Ativadoras de GTPase/genética , Leucócitos/fisiologia , Migração Transendotelial e Transepitelial , Proteínas Supressoras de Tumor/genética , Rigidez Vascular , Células Cultivadas , Proteínas Ativadoras de GTPase/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Leucócitos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Supressoras de Tumor/metabolismo
14.
Small GTPases ; 8(1): 1-15, 2017 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-27715453

RESUMO

Inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens. It serves as a protective response that involves leukocytes, blood vessels and molecular mediators with the purpose to eliminate the initial cause of cell injury and to initiate tissue repair. Inflammation is tightly regulated by the body and is associated with transient crossing of leukocytes through the blood vessel wall, a process called transendothelial migration (TEM) or diapedesis. TEM is a close collaboration between leukocytes on one hand and the endothelium on the other. Limiting vascular leakage during TEM but also when the leukocyte has crossed the endothelium is essential for maintaining vascular homeostasis. Although many details have been uncovered during the recent years, the molecular mechanisms from the vascular part that drive TEM still shows significant gaps in our understanding. This review will focus on the local signals that are induced in the endothelium that regulate leukocyte TEM and simultaneous preservation of endothelial barrier function.


Assuntos
Endotélio/metabolismo , Leucócitos/citologia , Migração Transendotelial e Transepitelial , Humanos , Transdução de Sinais
15.
Nat Commun ; 7: 10493, 2016 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-26814335

RESUMO

During immune surveillance and inflammation, leukocytes exit the vasculature through transient openings in the endothelium without causing plasma leakage. However, the exact mechanisms behind this intriguing phenomenon are still unknown. Here we report that maintenance of endothelial barrier integrity during leukocyte diapedesis requires local endothelial RhoA cycling. Endothelial RhoA depletion in vitro or Rho inhibition in vivo provokes neutrophil-induced vascular leakage that manifests during the physical movement of neutrophils through the endothelial layer. Local RhoA activation initiates the formation of contractile F-actin structures that surround emigrating neutrophils. These structures that surround neutrophil-induced endothelial pores prevent plasma leakage through actomyosin-based pore confinement. Mechanistically, we found that the initiation of RhoA activity involves ICAM-1 and the Rho GEFs Ect2 and LARG. In addition, regulation of actomyosin-based endothelial pore confinement involves ROCK2b, but not ROCK1. Thus, endothelial cells assemble RhoA-controlled contractile F-actin structures around endothelial pores that prevent vascular leakage during leukocyte extravasation.


Assuntos
Actinas/metabolismo , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Leucócitos/citologia , Transdução de Sinais , Migração Transendotelial e Transepitelial , Proteína rhoA de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/genética , Animais , Permeabilidade Capilar , Células Cultivadas , Endotélio Vascular/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Molécula 1 de Adesão Intercelular , Leucócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/citologia , Neutrófilos/metabolismo , Proteína rhoA de Ligação ao GTP/genética
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