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1.
Development ; 150(6)2023 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-36852644

RESUMO

Wt1 encodes a zinc finger protein that is crucial for epicardium development. Although WT1 is also expressed in coronary endothelial cells (ECs), the abnormal heart development observed in Wt1 knockout mice is mainly attributed to its functions in the epicardium. Here, we have generated an inducible endothelial-specific Wt1 knockout mouse model (Wt1KOΔEC). Deletion of Wt1 in ECs during coronary plexus formation impaired coronary blood vessels and myocardium development. RNA-Seq analysis of coronary ECs from Wt1KOΔEC mice demonstrated that deletion of Wt1 exerted a major impact on the molecular signature of coronary ECs and modified the expression of several genes that are dynamically modulated over the course of coronary EC development. Many of these differentially expressed genes are involved in cell proliferation, migration and differentiation of coronary ECs; consequently, the aforementioned processes were affected in Wt1KOΔEC mice. The requirement of WT1 in coronary ECs goes beyond the initial formation of the coronary plexus, as its later deletion results in defects in coronary artery formation. Through the characterization of these Wt1KOΔEC mouse models, we show that the deletion of Wt1 in ECs disrupts physiological blood vessel formation.


Assuntos
Vasos Coronários , Células Endoteliais , Camundongos , Animais , Células Endoteliais/metabolismo , Vasos Coronários/metabolismo , Pericárdio/metabolismo , Proliferação de Células/genética , Neovascularização Fisiológica/genética , Modelos Animais de Doenças , Camundongos Knockout , Miocárdio/metabolismo , Proteínas WT1/genética
2.
EMBO J ; 38(2)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30518533

RESUMO

Vascular endothelial growth factor receptor-3 (VEGFR3) signalling promotes lymphangiogenesis. While there are many reported mechanisms of VEGFR3 activation, there is little understanding of how VEGFR3 signalling is attenuated to prevent lymphatic vascular overgrowth and ensure proper lymph vessel development. Here, we show that endothelial cell-specific depletion of integrin-linked kinase (ILK) in mouse embryos hyper-activates VEGFR3 signalling and leads to overgrowth of the jugular lymph sacs/primordial thoracic ducts, oedema and embryonic lethality. Lymphatic endothelial cell (LEC)-specific deletion of Ilk in adult mice initiates lymphatic vascular expansion in different organs, including cornea, skin and myocardium. Knockdown of ILK in human LECs triggers VEGFR3 tyrosine phosphorylation and proliferation. ILK is further found to impede interactions between VEGFR3 and ß1 integrin in vitro and in vivo, and endothelial cell-specific deletion of an Itgb1 allele rescues the excessive lymphatic vascular growth observed upon ILK depletion. Finally, mechanical stimulation disrupts the assembly of ILK and ß1 integrin, releasing the integrin to enable its interaction with VEGFR3. Our data suggest that ILK facilitates mechanically regulated VEGFR3 signalling via controlling its interaction with ß1 integrin and thus ensures proper development of lymphatic vessels.


Assuntos
Integrina beta1/metabolismo , Linfangiogênese , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Vasos Linfáticos/citologia , Vasos Linfáticos/metabolismo , Camundongos , Fosforilação , Transdução de Sinais
3.
J Am Soc Nephrol ; 33(4): 786-808, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35260418

RESUMO

BACKGROUND: The cell-matrix adhesion between podocytes and the glomerular basement membrane is essential for the integrity of the kidney's filtration barrier. Despite increasing knowledge about the complexity of integrin adhesion complexes, an understanding of the regulation of these protein complexes in glomerular disease remains elusive. METHODS: We mapped the in vivo composition of the podocyte integrin adhesome. In addition, we analyzed conditional knockout mice targeting a gene (Parva) that encodes an actin-binding protein (α-parvin), and murine disease models. To evaluate podocytes in vivo, we used super-resolution microscopy, electron microscopy, multiplex immunofluorescence microscopy, and RNA sequencing. We performed functional analysis of CRISPR/Cas9-generated PARVA single knockout podocytes and PARVA and PARVB double knockout podocytes in three- and two-dimensional cultures using specific extracellular matrix ligands and micropatterns. RESULTS: We found that PARVA is essential to prevent podocyte foot process effacement, detachment from the glomerular basement membrane, and the development of FSGS. Through the use of in vitro and in vivo models, we identified an inherent PARVB-dependent compensatory module at podocyte integrin adhesion complexes, sustaining efficient mechanical linkage at the filtration barrier. Sequential genetic deletion of PARVA and PARVB induces a switch in structure and composition of integrin adhesion complexes. This redistribution of these complexes translates into a loss of the ventral actin cytoskeleton, decreased adhesion capacity, impaired mechanical resistance, and dysfunctional extracellular matrix assembly. CONCLUSIONS: The findings reveal adaptive mechanisms of podocyte integrin adhesion complexes, providing a conceptual framework for therapeutic strategies to prevent podocyte detachment in glomerular disease.


Assuntos
Barreira de Filtração Glomerular , Proteínas dos Microfilamentos , Podócitos , Animais , Barreira de Filtração Glomerular/metabolismo , Integrinas/metabolismo , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Podócitos/metabolismo
4.
Angiogenesis ; 25(2): 155-158, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35098411

RESUMO

Alpha-parvin (α-pv), an adaptor protein that mediates integrin-dependent cell-matrix interactions, is essential for endothelial cells migration and proliferation and is a key player in physiological angiogenesis. The role of α-pv in pathological angiogenesis is unknown. Here we demonstrate that endothelial α-pv is required for tumour angiogenesis. Using an inducible knockout approach in which the α-pv gene (Parva) was inactivated specifically in endothelial cells of brain tumour-bearing mice, we show that loss of endothelial α-pv results in reduced vessel density and decreased vascular complexity of the pathological neo-vasculature without affecting the structure of the brain vasculature around tumour. Reduced tumour vascularisation is associated with a significant increase in tumour cell apoptosis and a reduction in tumour volume. Together, our data show for the first time that endothelial α-pv is required for tumour vascularisation and tumour progression in vivo.


Assuntos
Células Endoteliais , Neoplasias , Animais , Apoptose/genética , Células Endoteliais/metabolismo , Camundongos , Neoplasias/patologia , Neovascularização Patológica/patologia , Neovascularização Fisiológica
5.
Circulation ; 140(13): 1100-1114, 2019 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-31401849

RESUMO

BACKGROUND: The incidence of acute cardiovascular complications is highly time-of-day dependent. However, the mechanisms driving rhythmicity of ischemic vascular events are unknown. Although enhanced numbers of leukocytes have been linked to an increased risk of cardiovascular complications, the role that rhythmic leukocyte adhesion plays in different vascular beds has not been studied. METHODS: We evaluated leukocyte recruitment in vivo by using real-time multichannel fluorescence intravital microscopy of a tumor necrosis factor-α-induced acute inflammation model in both murine arterial and venous macrovasculature and microvasculature. These approaches were complemented with genetic, surgical, and pharmacological ablation of sympathetic nerves or adrenergic receptors to assess their relevance for rhythmic leukocyte adhesion. In addition, we genetically targeted the key circadian clock gene Bmal1 (also known as Arntl) in a lineage-specific manner to dissect the importance of oscillations in leukocytes and components of the vessel wall in this process. RESULTS: In vivo quantitative imaging analyses of acute inflammation revealed a 24-hour rhythm in leukocyte recruitment to arteries and veins of the mouse macrovasculature and microvasculature. Unexpectedly, although in arteries leukocyte adhesion was highest in the morning, it peaked at night in veins. This phase shift was governed by a rhythmic microenvironment and a vessel type-specific oscillatory pattern in the expression of promigratory molecules. Differences in cell adhesion molecules and leukocyte adhesion were ablated when disrupting sympathetic nerves, demonstrating their critical role in this process and the importance of ß2-adrenergic receptor signaling. Loss of the core clock gene Bmal1 in leukocytes, endothelial cells, or arterial mural cells affected the oscillations in a vessel type-specific manner. Rhythmicity in the intravascular reactivity of adherent leukocytes resulted in increased interactions with platelets in the morning in arteries and in veins at night with a higher predisposition to acute thrombosis at different times as a consequence. CONCLUSIONS: Together, our findings point to an important and previously unrecognized role of artery-associated sympathetic innervation in governing rhythmicity in vascular inflammation in both arteries and veins and its potential implications in the occurrence of time-of-day-dependent vessel type-specific thrombotic events.


Assuntos
Artérias/imunologia , Endotélio Vascular/metabolismo , Inflamação/imunologia , Leucócitos/fisiologia , Trombose/fisiopatologia , Veias/imunologia , Animais , Artérias/inervação , Artérias/patologia , Adesão Celular , Células Cultivadas , Relógios Circadianos , Endotélio Vascular/patologia , Regulação da Expressão Gênica , Humanos , Microscopia Intravital , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Periodicidade , Receptores Adrenérgicos beta 2/metabolismo , Sistema Nervoso Simpático , Fator de Necrose Tumoral alfa/metabolismo , Veias/inervação , Veias/patologia
6.
Blood ; 130(7): 847-858, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28615221

RESUMO

Trafficking of polymorphonuclear neutrophils (PMNs) during inflammation critically depends on the ß2 integrins lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18) and macrophage-1 antigen (CD11b/CD18). Here, we identify coronin 1A (Coro1A) as a novel regulator of ß2 integrins that interacts with the cytoplasmic tail of CD18 and is crucial for induction of PMN adhesion and postadhesion events, including adhesion strengthening, spreading, and migration under flow conditions. Transition of PMN rolling to firm adhesion critically depends on Coro1A by regulating the accumulation of high-affinity LFA-1 in focal zones of adherent cells. Defective integrin affinity regulation in the genetic absence of Coro1A impairs leukocyte adhesion and extravasation in inflamed cremaster muscle venules in comparison with control animals. In a Helicobacter pylori mouse infection model, PMN infiltration into the gastric mucosa is dramatically reduced in Coro1A-/- mice, resulting in an attenuated gastric inflammation. Thus, Coro1A represents an important novel player in integrin biology, with key functions in PMN trafficking during innate immunity.


Assuntos
4-Butirolactona/análogos & derivados , Antígenos CD18/metabolismo , Movimento Celular , Imunidade Inata , Neutrófilos/citologia , Neutrófilos/metabolismo , 4-Butirolactona/metabolismo , Actinas/metabolismo , Animais , Sinalização do Cálcio , Adesão Celular , Gastrite/imunologia , Gastrite/microbiologia , Gastrite/patologia , Helicobacter pylori/fisiologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Antígeno de Macrófago 1/metabolismo , Camundongos Endogâmicos C57BL , Receptores Acoplados a Proteínas G/metabolismo , Reologia
7.
Arterioscler Thromb Vasc Biol ; 38(5): 1147-1158, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29567677

RESUMO

OBJECTIVE: During vascular development, integrin-mediated signaling regulates the formation and stabilization of cell-cell junctions, which are required for endothelial cell (EC) apical-basal polarity and proper deposition of the vascular basement membrane. Parvins are actin-binding proteins that facilitate the interaction of integrins with the actin cytoskeleton. The endothelium expresses 2 parvin isoforms: α-pv (α-parvin) and ß-pv (ß-parvin). Recently, we have shown that α-pv is critical for vessel growth and vessel stability at late embryonic developmental stages. The role of parvins during early embryonic development is unknown. APPROACH AND RESULTS: To investigate the role of endothelial parvins in the developing vasculature, we generated mice with ECs lacking both parvin isoforms by deleting α-pv in ECs in global ß-pv-/- mice (α-pvΔEC;ß-pv-/- mice). Here, we show that α-pvΔEC;ß-pv-/- mice die around embryonic day 11.5 and exhibit hemorrhages, immature capillary beds, and severe vascular defects in the central nervous system, including reduced vessel branching, increased vessel diameter, and balloon-like hemorrhagic clusters of ECs. Vessels in α-pvΔEC;ß-pv-/- embryos display disorganized cell-cell junctions, impaired endothelial apical-basal polarity, and discontinuous basement membranes. These vascular defects are accompanied by defective pericyte-vessel interaction. CONCLUSIONS: Our results show that parvins are critical for the organization of endothelial cell-cell junctions, the establishment of endothelial apical-basal polarity, and the integrity of the basement membrane.


Assuntos
Actinina/metabolismo , Vasos Sanguíneos/metabolismo , Polaridade Celular , Células Endoteliais/metabolismo , Junções Intercelulares/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neovascularização Fisiológica , Malformações Vasculares/metabolismo , Actinina/deficiência , Actinina/genética , Animais , Membrana Basal/metabolismo , Membrana Basal/patologia , Vasos Sanguíneos/embriologia , Forma Celular , Células Cultivadas , Células Endoteliais/patologia , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Junções Intercelulares/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Neovascularização Patológica , Pericitos/metabolismo , Pericitos/patologia , Transdução de Sinais , Malformações Vasculares/embriologia , Malformações Vasculares/genética
8.
Circ Res ; 117(1): 29-40, 2015 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-25925587

RESUMO

RATIONALE: Angiogenesis and vessel integrity depend on the adhesion of endothelial cells (ECs) to the extracellular matrix and to adjacent ECs. The focal adhesion protein α-parvin (α-pv) is essential for vascular development. However, the role of α-pv in ECs in vivo is not known. OBJECTIVE: To determine the function of α-pv in ECs during vascular development in vivo and the underlying mechanisms. METHODS AND RESULTS: We deleted the α-pv gene specifically in ECs of mice to study its role in angiogenesis and vascular development. Here, we show that endothelial-specific deletion of α-pv in mice results in late embryonic lethality associated with hemorrhages and reduced vascular density. Postnatal-induced EC-specific deletion of α-pv leads to retinal hypovascularization because of reduced vessel sprouting and excessive vessel regression. In the absence of α-pv, blood vessels display impaired VE-cadherin junction morphology. In vitro, α-pv-deficient ECs show reduced stable adherens junctions, decreased monolayer formation, and impaired motility, associated with reduced formation of integrin-mediated cell-extracellular matrix adhesion structures and an altered actin cytoskeleton. CONCLUSIONS: Endothelial α-pv is essential for vessel sprouting and for vessel stability.


Assuntos
Junções Aderentes/ultraestrutura , Vasos Sanguíneos/embriologia , Células Endoteliais/citologia , Endotélio Vascular/fisiologia , Proteínas dos Microfilamentos/fisiologia , Neovascularização Fisiológica/fisiologia , Junções Aderentes/fisiologia , Animais , Antígenos CD/análise , Vasos Sanguíneos/crescimento & desenvolvimento , Caderinas/análise , Movimento Celular , Forma Celular , Células Cultivadas , Citoesqueleto/ultraestrutura , Células Endoteliais/metabolismo , Endotélio Vascular/ultraestrutura , Matriz Extracelular/ultraestrutura , Feminino , Genes Letais , Células Endoteliais da Veia Umbilical Humana , Masculino , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Neovascularização Fisiológica/genética , Pseudópodes/fisiologia , Pseudópodes/ultraestrutura , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Vasos Retinianos/patologia
9.
EMBO J ; 29(2): 281-91, 2010 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-20033063

RESUMO

Dynamic interactions of cells with their environment regulate multiple aspects of tissue morphogenesis and function. Integrins are the major class of cell surface receptors that recognize and bind extracellular matrix proteins, resulting in the engagement and organization of the cytoskeleton as well as activation of signalling pathways to regulate cell behaviour and morphogenetic processes. The ternary complex of integrin-linked kinase (ILK), PINCH, and parvin (IPP complex), which was identified more than a decade ago, interacts with the cytoplasmic tail of beta integrins and couples them to the actin cytoskeleton. In addition, ILK has been shown to act as a serine/threonine kinase and to directly activate several signalling pathways downstream of integrins. However, the kinase activity of ILK and the precise functions of the IPP complex have remained elusive and controversial. This review focuses on the recent advances made towards understanding the specialized roles this complex and its individual components have acquired during evolution.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação a DNA/genética , Humanos , Proteínas dos Microfilamentos/genética , Dados de Sequência Molecular , Morfogênese , Proteínas Serina-Treonina Quinases/genética , Alinhamento de Sequência
10.
J Cell Sci ; 125(Pt 21): 5233-40, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22946061

RESUMO

The focal adhesion (FA) protein PINCH-1 is required for the survival of primitive endoderm (PrE) cells. How PINCH-1 regulates this fundamental process is not known. Here, we use embryoid bodies (EBs) and isolated EB-derived PrE cells to investigate the mechanisms by which PINCH-1 promotes PrE survival. We report that loss of PINCH-1 in PrE cells leads to a sustained activity of JNK and the pro-apoptotic factor Bax. Mechanistically, the sustained JNK activation was due to diminished levels of the JNK inhibitory factor Ras suppressor protein-1 (RSU-1), whose stability was severely reduced upon loss of PINCH-1. Chemical inhibition of JNK attenuated apoptosis of PrE cells but failed to reduce Bax activity. The increased Bax activity was associated with reduced integrin signalling and diminished Bcl-2 levels, which were shown to inhibit Bax. Altogether our findings show that PINCH-1 is a pro-survival factor that prevents apoptosis of PrE cells by modulating two independent signalling pathways; PINCH-1 inhibits JNK-mediated apoptosis by stabilising the PINCH-1 binding protein RSU-1 and promotes Bcl-2-dependent pro-survival signalling downstream of integrins.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Endoderma/citologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Endoderma/metabolismo , Ativação Enzimática , Deleção de Genes , Integrinas/metabolismo , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Estabilidade Proteica , Fatores de Transcrição/metabolismo
11.
JCI Insight ; 9(5)2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38300714

RESUMO

TAR DNA-binding protein 43 (TDP-43) is a DNA/RNA-binding protein that regulates gene expression, and its malfunction in neurons has been causally associated with multiple neurodegenerative disorders. Although progress has been made in understanding the functions of TDP-43 in neurons, little is known about its roles in endothelial cells (ECs), angiogenesis, and vascular function. Using inducible EC-specific TDP-43-KO mice, we showed that TDP-43 is required for sprouting angiogenesis, vascular barrier integrity, and blood vessel stability. Postnatal EC-specific deletion of TDP-43 led to retinal hypovascularization due to defects in vessel sprouting associated with reduced EC proliferation and migration. In mature blood vessels, loss of TDP-43 disrupted the blood-brain barrier and triggered vascular degeneration. These vascular defects were associated with an inflammatory response in the CNS with activation of microglia and astrocytes. Mechanistically, deletion of TDP-43 disrupted the fibronectin matrix around sprouting vessels and reduced ß-catenin signaling in ECs. Together, our results indicate that TDP-43 is essential for the formation of a stable and mature vasculature.


Assuntos
Células Endoteliais , Doenças Neuroinflamatórias , Camundongos , Animais , Células Endoteliais/metabolismo , Angiogênese , Neovascularização Fisiológica/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo
12.
EMBO J ; 28(20): 3132-44, 2009 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-19798050

RESUMO

During blood vessel development, vascular smooth muscle cells (vSMCs) and pericytes (PCs) are recruited to nascent vessels to stabilize them and to guide further vessel remodelling. Here, we show that loss of the focal adhesion (FA) protein alpha-parvin (alpha-pv) in mice leads to embryonic lethality due to severe cardiovascular defects. The vascular abnormalities are characterized by poor vessel remodelling, impaired coverage of endothelial tubes with vSMC/PCs and defective association of the recruited vSMC/PCs with endothelial cells (ECs). Alpha-pv-deficient vSMCs are round and hypercontractile leading either to their accumulation in the tissue or to local vessel constrictions. Because of the high contractility, alpha-pv-deficient vSMCs fail to polarize their cytoskeleton resulting in loss of persistent and directed migration. Mechanistically, the absence of alpha-pv leads to increased RhoA and Rho-kinase (ROCK)-mediated signalling, activation of myosin II and actomyosin hypercontraction in vSMCs. Our findings show that alpha-pv represents an essential adhesion checkpoint that controls RhoA/ROCK-mediated contractility in vSMCs.


Assuntos
Vasos Sanguíneos/citologia , Movimento Celular/fisiologia , Proteínas dos Microfilamentos/fisiologia , Músculo Liso Vascular/citologia , Transdução de Sinais/fisiologia , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Doenças Cardiovasculares/embriologia , Doenças Cardiovasculares/genética , Movimento Celular/genética , Eletroforese em Gel de Poliacrilamida , Células Endoteliais/citologia , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Proteínas dos Microfilamentos/genética , Pericitos/citologia , RNA Interferente Pequeno , Transdução de Sinais/genética
13.
Front Cell Dev Biol ; 11: 1169962, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37384248

RESUMO

Aggregation of the Tar DNA-binding protein of 43 kDa (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia and likely contributes to disease by loss of nuclear function. Analysis of TDP-43 function in knockout zebrafish identified an endothelial directional migration and hypersprouting phenotype during development prior lethality. In human umbilical vein cells (HUVEC) the loss of TDP-43 leads to hyperbranching. We identified elevated expression of FIBRONECTIN 1 (FN1), the VASCULAR CELL ADHESION MOLECULE 1 (VCAM1), as well as their receptor INTEGRIN α4ß1 (ITGA4B1) in HUVEC cells. Importantly, reducing the levels of ITGA4, FN1, and VCAM1 homologues in the TDP-43 loss-of-function zebrafish rescues the angiogenic defects indicating the conservation of human and zebrafish TDP-43 function during angiogenesis. Our study identifies a novel pathway regulated by TDP-43 important for angiogenesis during development.

14.
Curr Top Dev Biol ; 149: 203-261, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35606057

RESUMO

Hematopoietic stem cells (HSCs), the apex of the hierarchically organized blood cell production system, are generated in the yolk sac, aorta-gonad-mesonephros region and placenta of the developing embryo. To maintain life-long hematopoiesis, HSCs emigrate from their site of origin and seed in distinct microenvironments, called niches, of fetal liver and bone marrow where they receive supportive signals for self-renewal, expansion and production of hematopoietic progenitor cells (HPCs), which in turn orchestrate the production of the hematopoietic effector cells. The interactions of hematopoietic stem and progenitor cells (HSPCs) with niche components are to a large part mediated by the integrin superfamily of adhesion molecules. Here, we summarize the current knowledge regarding the functional properties of integrins and their activators, Talin-1 and Kindlin-3, for HSPC generation, function and fate decisions during development and in adulthood. In addition, we discuss integrin-mediated mechanosensing for HSC-niche interactions, ex vivo protocols aimed at expanding HSCs for therapeutic use, and recent approaches targeting the integrin-mediated adhesion in leukemia-inducing HSCs in their protecting, malignant niches.


Assuntos
Células-Tronco Hematopoéticas , Integrinas , Medula Óssea , Feminino , Hematopoese , Humanos , Integrinas/metabolismo , Mesonefro , Gravidez
15.
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
16.
Cell Syst ; 12(3): 248-262.e7, 2021 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-33592194

RESUMO

Aggressive brain tumors like glioblastoma depend on support by their local environment and subsets of tumor parenchymal cells may promote specific phases of disease progression. We investigated the glioblastoma microenvironment with transgenic lineage-tracing models, intravital imaging, single-cell transcriptomics, immunofluorescence analysis as well as histopathology and characterized a previously unacknowledged population of tumor-associated cells with a myeloid-like expression profile (TAMEP) that transiently appeared during glioblastoma growth. TAMEP of mice and humans were identified with specific markers. Notably, TAMEP did not derive from microglia or peripheral monocytes but were generated by a fraction of CNS-resident, SOX2-positive progenitors. Abrogation of this progenitor cell population, by conditional Sox2-knockout, drastically reduced glioblastoma vascularization and size. Hence, TAMEP emerge as a tumor parenchymal component with a strong impact on glioblastoma progression.


Assuntos
Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/patologia , Glioblastoma/irrigação sanguínea , Glioblastoma/patologia , Células Mieloides/patologia , Animais , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Progressão da Doença , Humanos , Masculino , Camundongos , Tecido Parenquimatoso/irrigação sanguínea , Tecido Parenquimatoso/patologia
17.
PLoS One ; 15(3): e0230380, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32163511

RESUMO

Epidermal morphogenesis and hair follicle (HF) development depend on the ability of keratinocytes to adhere to the basement membrane (BM) and migrate along the extracellular matrix. Integrins are cell-matrix receptors that control keratinocyte adhesion and migration, and are recognized as major regulators of epidermal homeostasis. How integrins regulate the behavior of keratinocytes during epidermal morphogenesis remains insufficiently understood. Here, we show that α-parvin (α-pv), a focal adhesion protein that couples integrins to actin cytoskeleton, is indispensable for epidermal morphogenesis and HF development. Inactivation of the murine α-pv gene in basal keratinocytes results in keratinocyte-BM detachment, epidermal thickening, ectopic keratinocyte proliferation and altered actin cytoskeleton polarization. In vitro, α-pv-null keratinocytes display reduced adhesion to BM matrix components, aberrant spreading and stress fibers formation, and impaired directed migration. Together, our data demonstrate that α-pv controls epidermal homeostasis by facilitating integrin-mediated adhesion and actin cytoskeleton organization in keratinocytes.


Assuntos
Membrana Basal/metabolismo , Epiderme/crescimento & desenvolvimento , Folículo Piloso/metabolismo , Queratinócitos/metabolismo , Proteínas dos Microfilamentos/fisiologia , Morfogênese/fisiologia , Actinas/metabolismo , Animais , Membrana Basal/citologia , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Adesões Focais/metabolismo , Integrinas/metabolismo , Queratinócitos/citologia , Camundongos , Camundongos Transgênicos
18.
Front Cell Dev Biol ; 8: 708, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32850828

RESUMO

Development and homeostasis of blood vessels critically depend on the regulation of endothelial cell-cell junctions. VE-cadherin (VEcad)-based cell-cell junctions are connected to the actin cytoskeleton and regulated by actin-binding proteins. Coronin 1B (Coro1B) is an actin binding protein that controls actin networks at classical lamellipodia. The role of Coro1B in endothelial cells (ECs) is not fully understood and investigated in this study. Here, we demonstrate that Coro1B is a novel component and regulator of cell-cell junctions in ECs. Immunofluorescence studies show that Coro1B colocalizes with VEcad at cell-cell junctions in monolayers of ECs. Live-cell imaging reveals that Coro1B is recruited to, and operated at actin-driven membrane protrusions at cell-cell junctions. Coro1B is recruited to cell-cell junctions via a mechanism that requires the relaxation of the actomyosin cytoskeleton. By analyzing the Coro1B interactome, we identify integrin-linked kinase (ILK) as new Coro1B-associated protein. Coro1B colocalizes with α-parvin, an interactor of ILK, at the leading edge of lamellipodia protrusions. Functional experiments reveal that depletion of Coro1B causes defects in the actin cytoskeleton and cell-cell junctions. Finally, in matrigel tube network assays, depletion of Coro1B results in reduced network complexity, tube number and tube length. Together, our findings point toward a critical role for Coro1B in the dynamic remodeling of endothelial cell-cell junctions and the assembly of endothelial networks.

19.
Nat Commun ; 10(1): 5243, 2019 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-31748531

RESUMO

Familial exudative vitreoretinopathy (FEVR) is a human disease characterized by defective retinal angiogenesis and associated complications that can result in vision loss. Defective Wnt/ß-catenin signaling is an established cause of FEVR, whereas other molecular alterations contributing to the disease remain insufficiently understood. Here, we show that integrin-linked kinase (ILK), a mediator of cell-matrix interactions, is indispensable for retinal angiogenesis. Inactivation of the murine Ilk gene in postnatal endothelial cells results in sprouting defects, reduced endothelial proliferation and disruption of the blood-retina barrier, resembling phenotypes seen in established mouse models of FEVR. Retinal vascularization defects are phenocopied by inducible inactivation of the gene for α-parvin (Parva), an interactor of ILK. Screening genomic DNA samples from exudative vitreoretinopathy patients identifies three distinct mutations in human ILK, which compromise the function of the gene product in vitro. Together, our data suggest that defective cell-matrix interactions are linked to Wnt signaling and FEVR.


Assuntos
Barreira Hematorretiniana/metabolismo , Células Endoteliais/metabolismo , Vitreorretinopatias Exsudativas Familiares/genética , Neovascularização Fisiológica/genética , Proteínas Serina-Treonina Quinases/genética , Vasos Retinianos/crescimento & desenvolvimento , Animais , Células Endoteliais/citologia , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Proteínas dos Microfilamentos/genética , Fenótipo , Via de Sinalização Wnt/genética
20.
Methods Enzymol ; 426: 239-89, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17697888

RESUMO

Integrins mediate cell adhesion, permit traction forces important for cell migration, and cross-talk with growth factor receptors to regulate cell proliferation, cell survival, and cell differentiation. The plethora of functions explains their central role for development and disease. The progress in mouse genetics and the ease with which the mouse genome can be manipulated enormously contributed to our understanding of how integrins exert their functions at the molecular level. In the present chapter, we describe tests that are routinely used in our laboratory to investigate embryos, organs, and cells (peri-implantation embryos, hematopoietic system, epidermis, and hair follicles) that lack the expression of integrins or integrin-associated proteins.


Assuntos
Blastocisto/fisiologia , Embrião de Mamíferos/citologia , Sistema Hematopoético/embriologia , Integrinas/fisiologia , Pele/embriologia , Animais , Células Cultivadas , Embrião de Mamíferos/anatomia & histologia , Feminino , Queratinócitos/citologia , Queratinócitos/fisiologia , Camundongos , Gravidez , Pele/citologia
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