RESUMO
To determine whether leukocytes need to open endothelial cell contacts during extravasation, we decided to generate mice with strongly stabilized endothelial junctions. To this end, we replaced VE-cadherin genetically by a VE-cadherin-α-catenin fusion construct. Such mice were completely resistant to the induction of vascular leaks by VEGF or histamine. Neutrophil or lymphocyte recruitment into inflamed cremaster, lung and skin were strongly inhibited in these mice, documenting the importance of the junctional route in vivo. Surprisingly, lymphocyte homing into lymph nodes was not inhibited. VE-cadherin-α-catenin associated more intensely with the actin cytoskeleton as demonstrated by its membrane mobility and detergent extractability. Our results establish the junctional route as the main pathway for extravasating leukocytes in several, although not in all tissues. Furthermore, in these tissues, plasticity of the VE-cadherin-catenin complex is central for the leukocyte diapedesis mechanism.
Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Permeabilidade Capilar , Leucócitos/fisiologia , Migração Transendotelial e Transepitelial , alfa Catenina/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Antígenos CD/genética , Caderinas/genética , Células Cultivadas , Endotélio Vascular/metabolismo , Feminino , Técnicas de Introdução de Genes , Inflamação/genética , Inflamação/metabolismo , Junções Intercelulares/metabolismo , Leucócitos/metabolismo , Pulmão/metabolismo , Linfonodos/metabolismo , Masculino , Camundongos , Músculo Esquelético/metabolismo , Pele/metabolismo , alfa Catenina/genéticaRESUMO
The endothelial layer of blood vessels controls the passage of cells and solutes from the blood into the surrounding tissue. Crucial for this regulation is the integrity of endothelial cell-cell junctions. Various molecular mechanisms control junctional integrity of the endothelial layer including GTPases, modulation of the actomyosin cytoskeleton and phosphorylation and dephosphorylation of junctional proteins. Several kinases and phosphatases have been identified that are good candidates for the regulation of the endothelial barrier function. For some of them, in vivo evidence has recently been presented that highlights their importance in either the regulation of vascular permeability or leukocyte extravasation. This review will summarize current knowledge about the regulation of endothelial junctions by kinases and phosphatases. In particular, the role of the endothelial specific phosphatase VE-PTP in the context of endothelial cell contact stability will be highlighted.
Assuntos
Células Endoteliais/enzimologia , Endotélio Vascular/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases/metabolismo , Animais , Humanos , FosforilaçãoRESUMO
Endothelial cells (ECs) display remarkable plasticity during development before becoming quiescent and functionally mature. EC maturation is directed by several known transcription factors (TFs), but the specific set of TFs responsible for promoting high-resistance barriers, such as the blood-brain barrier (BBB), have not yet been fully defined. Using expression mRNA data from published studies on ex vivo ECs from the central nervous system (CNS), we predicted TFs that induce high-resistance barrier properties of ECs as in the BBB. We used our previously established method to generate ECs from human pluripotent stem cells (hPSCs), and then we overexpressed the candidate TFs in hPSC-ECs and measured barrier resistance and integrity using electric cell-substrate impedance sensing, trans-endothelial electrical resistance and FITC-dextran permeability assays. SOX18 and TAL1 were the strongest EC barrier-inducing TFs, upregulating Wnt-related signaling and EC junctional gene expression, respectively, and downregulating EC proliferation-related genes. These TFs were combined with SOX7 and ETS1 that together effectively induced EC barrier resistance, decreased paracellular transport and increased protein expression of tight junctions and induce mRNA expression of several genes involved in the formation of EC barrier and transport. Our data shows identification of a transcriptional network that controls barrier resistance in ECs. Collectively this data may lead to novel approaches for generation of in vitro models of the BBB.
Assuntos
Células Endoteliais/metabolismo , Fatores de Transcrição/metabolismo , Barreira Hematoencefálica/citologia , Diferenciação Celular , Células Endoteliais/citologia , Humanos , Células-Tronco Pluripotentes/citologiaRESUMO
Vascular endothelial (VE)-protein tyrosine phosphatase (PTP) associates with VE-cadherin, thereby supporting its adhesive activity and endothelial junction integrity. VE-PTP also associates with Tie-2, dampening the tyrosine kinase activity of this receptor that can support stabilization of endothelial junctions. Here, we have analyzed how interference with VE-PTP affects the stability of endothelial junctions in vivo. Blocking VE-PTP by antibodies, a specific pharmacological inhibitor (AKB-9778), and gene ablation counteracted vascular leak induction by inflammatory mediators. In addition, leukocyte transmigration through the endothelial barrier was attenuated. Interference with Tie-2 expression in vivo reversed junction-stabilizing effects of AKB-9778 into junction-destabilizing effects. Furthermore, lack of Tie-2 was sufficient to weaken the vessel barrier. Mechanistically, inhibition of VE-PTP stabilized endothelial junctions via Tie-2, which triggered activation of Rap1, which then caused the dissolution of radial stress fibers via Rac1 and suppression of nonmuscle myosin II. Remarkably, VE-cadherin gene ablation did not abolish the junction-stabilizing effect of the VE-PTP inhibitor. Collectively, we conclude that inhibition of VE-PTP stabilizes challenged endothelial junctions in vivo via Tie-2 by a VE-cadherin-independent mechanism. In the absence of Tie-2, however, VE-PTP inhibition destabilizes endothelial barrier integrity in agreement with the VE-cadherin-supportive effect of VE-PTP.
Assuntos
Caderinas/deficiência , Células Endoteliais/metabolismo , Receptor TIE-2/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Compostos de Anilina/farmacologia , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/ultraestrutura , Deleção de Genes , Inativação Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Ácidos Sulfônicos/farmacologia , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
The passage of leukocytes across the blood vessel wall is a fundamental event in the inflammatory response. During the last decades, there has been significant progress in understanding the molecular mechanisms involved in leukocyte transmigration. However, it is still a matter of debate whether leukocytes migrate paracellularly or transcellularly through an endothelial cell layer. We could recently show that a VE-cadherin-α-catenin fusion protein locks endothelial junctions in the skin and strongly reduces leukocyte diapedesis in lung, skin and cremaster, establishing the paracellular route as the major transmigration pathway in these tissues. However, the homing of naïve lymphocytes into lymph nodes and extravasation of neutrophils in the inflamed peritoneum were not affected by VE-cadherin-α-catenin. This unexpected heterogeneity of the diapedesis process in different tissues as well as the complexity and dynamics of the cadherin-catenin complex in regulating endothelial junctions will be discussed.