Visualization of Endothelial Actin Cytoskeleton in the Mouse Retina

Angiogenesis requires coordinated changes in cell shape of endothelial cells (ECs), orchestrated by the actin cytoskeleton. The mechanisms that regulate this rearrangement in vivo are poorly understood - largely because of the difficulty to visualize filamentous actin (F-actin) structures with sufficient resolution. Here, we use transgenic mice expressing Lifeact-EGFP to visualize F-actin in ECs. We show that in the retina, Lifeact-EGFP expression is largely restricted to ECs allowing detailed visualization of F-actin in ECs in situ. Lifeact-EGFP labels actin associated with cell-cell junctions, apical and basal membranes and highlights actin-based structures such as filopodia and stress fiber-like cytoplasmic bundles. We also show that in the skin and the skeletal muscle, Lifeact-EGFP is highly expressed in vascular mural cells (vMCs), enabling vMC imaging. In summary, our results indicate that the Lifeact-EGFP transgenic mouse in combination with the postnatal retinal angiogenic model constitutes an excellent system for vascular cell biology research. Our approach is ideally suited to address structural and mechanistic details of angiogenic processes, such as endothelial tip cell migration and fusion, EC polarization or lumen formation

Integrin-linked kinase controls retinal angiogenesis and is linked to wnt signaling and exudative vitreoretinopathy

Familial exudative vitreoretinopathy (FEVR) is a human disease characterized by defective retinal angiogenesis and associated complications that can result in vision loss. Defective Wnt/β-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

F-actin-rich contractile endothelial pores prevent vascular leakage during leukocyte diapedesis through local RhoA signalling

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

Loss of TDP-43 causes ectopic endothelial sprouting and migration defects through increased fibronectin, vcam 1 and integrin α4/β1

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 α4β1 (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

Parvins Are Required for Endothelial Cell–Cell Junctions and Cell Polarity During Embryonic Blood Vessel Formation

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: -pv (-parvin) and -pv (-parvin). Recently, we have shown that -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 -pv in ECs in global -pv(-/-) mice (-pv(EC);-pv(-/-) mice). Here, we show that -pv(EC);-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 -pv(EC);-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

The ILK/PINCH/parvin complex: the kinase is dead, long live the pseudokinase!

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 β 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

α-parvin controls vascular mural cell recruitment to vessel wall by regulating RhoA/ROCK signalling

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 α-parvin (α-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). α-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, α-pv-deficient vSMCs fail to polarize their cytoskeleton resulting in loss of persistent and directed migration. Mechanistically, the absence of α-pv leads to increased RhoA and Rho-kinase (ROCK)-mediated signalling, activation of myosin II and actomyosin hypercontraction in vSMCs. Our findings show that α-pv represents an essential adhesion checkpoint that controls RhoA/ROCK-mediated contractility in vSMCs

VEGF-A/Notch-Induced Podosomes Proteolyse Basement Membrane Collagen-IV during Retinal Sprouting Angiogenesis

International audienceDuring angiogenic sprouting, endothelial tip cells emerge from existing vessels in a process that requires vascular basement membrane degradation. Here, we show that F-actin/cortactin/P-Src-based matrix-degrading microdomains called podosomes contribute to this step. In vitro, VEGF-A/Notch signaling regulates the formation of functional podosomes in endothelial cells. Using a retinal neovascularization model, we demonstrate that tip cells assemble podosomes during physiological angiogenesis in vivo. In the retina, podosomes are also part of an interconnected network that surrounds large microvessels and impinges on the underlying basement membrane. Consistently, collagen-IV is scarce in podosome areas. Moreover, Notch inhibition exacerbates podosome formation and collagen-IV loss. We propose that the localized proteolytic action of podosomes on basement membrane collagen-IV facilitates endothelial cell sprouting and anastomosis within the developing vasculature. The identification of podosomes as key components of the sprouting machinery provides another opportunity to target angiogenesis therapeutically

Integrin-linked kinase controls retinal angiogenesis and is linked to Wnt signaling and exudative vitreoretinopathy

Familial exudative vitreoretinopathy (FEVR) is a human disease characterized by defective retinal angiogenesis and associated complications that can result in vision loss. Defective Wnt/β-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