Biology subjects

Lagendijk, A. K.

Publications and source records attributed to Lagendijk, A. K..

9 recordsLinked to original sources

bioRxiv2025

Integrin β1-Talin1 at focal adhesions underpin uncontrolled endothelial cell enlargement in live cerebral cavernous malformation vasculature

Cerebral cavernous malformations (CCMs) are vascular anomalies caused by loss of CCM gene function and consequent hyperactivation of MEKK3-KLF2/4 signaling in endothelial cells. Excess integrin {beta}1 activity has been associated with lesion growth, yet the precise mechanistic and biomechanical roles of the integrin-KLF2/4 hierarchy in CCM cellular pathogenesis are not fully understood. Using live imaging of endothelial Vinculin in Ccm1-deficient zebrafish, we demonstrate excessive, mechanically active focal adhesions in an in vivo model of CCM pathology. We validate this in CCM1-deficient endothelial cells and show a redistribution of mechanical tension from cell-cell junctions to focal adhesions. Genetic deletion of Talin1 to decouple focal adhesions from the cell cortex and inhibit integrin {beta}1 signaling demonstrates the integrin {beta}1-Talin1 complex is essential for vascular malformations in ccm1 mutants by driving endothelial cell enlargement. We show integrin {beta}1-Talin1 act independent or downstream of KLF2/4, rather than upstream as previously suggested. Thus, we reposition the role of integrin {beta}1-Talin1 in CCM pathogenesis, demonstrating an essential function in driving cell enlargement, which underpins lesion growth.

cell biology↗

bioRxiv2024

Circumferential actomyosin bundles drive endothelial cell deformations to constrict blood vessels

Following the formation of new blood vessels, vascular remodelling ensues to generate a hierarchical network of vascular tubes with optimal connections and diameters for efficient blood perfusion of tissues. How transitions in endothelial cell (EC) number and shape are coordinated to define vessel diameter during development remains an open question. In this study, we discovered EC deformations, rearrangements and transient formation of self-seam junctions as key mechanisms that explain a negative relationship between cell number and vessel diameter. High-resolution analysis of actin cytoskeleton organization disclosed the generation of tension-bearing, circumferential actomyosin bundles in the endothelial cortex that drive EC deformation and vessel constriction. Importantly, the loss of circumferential actin bundles in krit1/ccm1-deficient ECs causes cell enlargement and impaired vessel constriction that culminate in dilated vessels, characteristic of cerebral cavernous malformation. Our multiscale study therefore underpins circumferential actomyosin-driven EC deformations in controlling vessel size and in the prevention of vascular malformations.

cell biology↗

bioRxiv2023

Cerebral Cavernous Malformation severity is impacted by distinct forms of Hyaluronic acid in the vascular microenvironment

Cerebral Cavernous Malformations (CCMs) are vascular lesions that predominantly form in blood vessels of the central nervous system (CNS) upon loss of the CCM multimeric protein complex. The endothelial cells (ECs) within CCM lesions are characterised by overactive MEKK3 kinase and KLF2/4 transcription factor signalling, leading to pathological changes such as increased EC spreading and reduced junctional integrity. Concomitant to aberrant EC signalling, non-autonomous signals from the extracellular matrix (ECM) have also been implicated in CCM lesion growth and these factors might explain why CCM lesions mainly develop in the CNS. Here, we adapted a three dimensional (3D) microfluidic system to examine CCM1 deficient human micro-vessels in distinctive ECMs. We validate that EC pathological hallmarks are maintained in this 3D model. We further show that key genes responsible for homeostasis of Hyaluronic Acid (HA), a major ECM component of the CNS, are dysregulated in CCM. Supplementing the ECM in our model with forms of HA that are predicted to be reduced, inhibits CCM cellular phenotypes, independent of KLF2/4. This study thereby provides a proof-of-principle that ECM embedded 3D microfluidic models are ideally suited to identify how changes in ECM structure and signalling impact vascular malformations.

cell biology↗

bioRxiv2022

ACE2 is necessary for SARS-CoV-2 infection and sensing by macrophages but not sufficient for productive viral replication

Macrophages are key cellular contributors to COVID-19 pathogenesis. Whether SARS-CoV-2 can enter macrophages, replicate and release new viral progeny remains controversial. Similarly, whether macrophages need to sense replicating virus to drive cytokine release is also unclear. Macrophages are heterogeneous cells poised to respond to their local microenvironment, and accordingly, the SARS-CoV-2 entry receptor ACE2 is only present on a subset of macrophages at sites of human infection. Here, we use in vitro approaches to investigate how SARS-CoV-2 interacts with ACE2-negative and ACE2-positive human macrophages and determine how these macrophage populations sense and respond to SARS-CoV-2. We show that SARS-CoV-2 does not replicate within ACE2-negative human macrophages and does not induce pro-inflammatory cytokine expression. By contrast, ACE2 expression in human macrophages permits SARS-CoV-2 entry, replication, and virion release. ACE2-expressing macrophages sense replicating virus to trigger pro-inflammatory and anti-viral programs that limit virus release. These combined findings resolve several controversies regarding macrophage-SARS-CoV-2 interactions and identify a signaling circuit by which macrophages sense SARS-CoV-2 cell entry and respond by restricting viral replication. One sentence summaryLack of macrophage ACE2 expression precludes SARS-CoV-2 entry and sensing, while ACE2-expressing macrophages sense intramacrophage SARS-CoV-2 replication to induce rapid anti-viral responses that limit new virion release.

immunology↗

bioRxiv2022

Dynamically regulated Focal adhesions coordinate endothelial cell remodelling in developing vasculature

The assembly of a mature vascular network involves the coordinated control of cell shape changes to regulate morphogenesis of a complex vascular network. Cellular changes include a process of endothelial cell (EC) elongation which is essential for establishing appropriately sized lumens during vessel maturation1-3. However how EC elongation is dynamically regulated in vivo is not fully understood since live monitoring of this event can be challenging in animal models. Here, we utilise the live imaging capacity of the zebrafish to explore how integrin adhesion complexes, known as Focal Adhesions (FAs), control EC dynamics in live flow pressured vasculature. To do this, we generated a zebrafish mutant, deficient for the integrin adaptor protein Talin1. Notably, unlike the severe cardiovascular defects that arise in Talin1 knockout mice4, vasculogenesis still occurs normally talin1 mutants and cardiac output remains sufficient up to two days post fertilisation (dpf). This allowed us to uncouple primary roles for FAs in ECs during subsequent morphogenesis events, including angiogenesis and vessel remodelling, without interference of secondary effects that might occur due to systemic vessel failure or loss of blood flow. We further established a FA marker line, expressing endothelial Vinculin-eGFP, and demonstrated that FAs are lost in our talin1 mutants. This Vinculin transgene represents the first in vivo model to monitor endothelial FA dynamics. Loss of FAs in talin1 mutants, leads to compromised F-actin rearrangements, which perturb EC elongation and cell-cell junction linearisation during vessel remodelling. Chemical induction of actin polymerisation can restore these cellular phenotypes, suggesting a recovery of actin rearrangements that are sufficient to allow cell and junction shape changes. Together, we have identified that FAs are essential for active guidance of EC elongation and junction linearisation in flow pressured vessels. These observations can explain the severely compromised vessel beds, haemorrhage and vascular leakage that has been observed in mouse models that lack integrin signalling4-8.

cell biology↗

bioRxiv2021

Pkd1 and Wnt5a genetically interact to control lymphatic vascular morphogenesis in mice

Lymphatic vascular development is regulated by well-characterised signalling and transcriptional pathways. These pathways regulate lymphatic endothelial cell (LEC) migration, motility, polarity and and morphogenesis. Canonical and non-canonical WNT signalling pathways are known to control LEC polarity and development of lymphatic vessels and valves. PKD1, encoding Polycystin-1, is the most commonly mutated gene in polycystic kidney disease but has also been shown to be essential in lymphatic vascular morphogenesis. The mechanism by which Pkd1 acts during lymphangiogenesis remains unclear. Here we find that loss of non-canonical WNT signalling components Wnt5a and Ryk phenocopy lymphatic defects seen in Pkd1 knockout mice. To investigate genetic interaction, we generated Pkd1/Wnt5a double knockout mice. Loss of Wnt5a suppressed phenotypes seen in the lymphatic vasculature of Pkd1-/- mice and Pkd1 deletion suppressed phenotypes observed in Wnt5a-/- mice. Thus, we report mutually suppressive roles for Pkd1 and Wnt5a, with developing lymphatic networks restored to a more wild-type state in double mutant mice. This genetic interaction between Pkd1 and the non-canonical WNT signalling pathway ultimately controls LEC polarity and the morphogenesis of developing vessel networks. Our work suggests that Pkd1 acts at least in part by regulating non-canonical WNT signalling during the formation of lymphatic vascular networks.

developmental biology↗

bioRxiv2021

Endothelial cells elicit a pro-inflammatory response to SARS-COV-2 without productive viral infection

ObjectivesThrombotic 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. MethodsHere, we use patient autopsy samples, primary human endothelial cells and an in vitro model of the pulmonary epithelial-endothelial cell barrier to show that primary human endothelial cells express very low levels the SARS-CoV-2 receptor ACE2 and the protease TMPRSS2. ResultsAccordingly, endothelial cells can only be infected when SARS-CoV-2 is present at very high concentrations. However, this is not a productive infection (i.e. no infectious virus is produced) and viral entry induces an inflammatory response. 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. They do however sense and respond to infection in the adjacent epithelial cells, increasing ICAM-1 expression and releasing pro-inflammatory cytokines. ConclusionsTaken together, these data suggest that in vivo, endothelial cells are unlikely to be infected with SARS-CoV-2 and that infection is only likely to 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 are still likely to play a key role in SARS-CoV-2 pathogenesis by sensing adjacent infection and mounting a pro-inflammatory response to SARS-CoV-2.

cell biology↗

bioRxiv2020

Blood vessel occlusion by Cryptococcus neoformans is a mechanism for hemorrhagic dissemination of infection

Meningitis caused by infectious pathogens are associated with vessel damage and infarct formation, however the physiological cause is unknown. Cryptococcus neoformans, is a human fungal pathogen and causative agent of cryptococcal meningitis, where vascular events are observed in up to 30% of cases, predominantly in severe infection. Therefore, we aimed to investigate how infection may lead to vessel damage and associated pathogen dissemination using a zebrafish model for in vivo live imaging. We find that cryptococcal cells become trapped within the vasculature (dependent on there size) and proliferate there resulting in vasodilation. Localised cryptococcal growth, originating from a single or small number of cryptococcal cells in the vasculature was associated with sites of dissemination and simultaneously with loss of blood vessel integrity. Using a cell-cell junction tension reporter we identified dissemination from intact blood vessels and where vessel rupture occurred. Finally, we manipulated blood vessel stifness via cell junctions and found increased stiffness resulted in increased dissemination. Therefore, global vascular vasodilation occurs following infection, resulting in increased vessel tension which subsequently increases dissemination events, representing a positive feedback loop. Thus, we identify a mechanism for blood vessel damage during cryptococcal infection that may represent a cause of vascular damage and cortical infarction more generally in infective meningitis.

pathology↗

bioRxiv2017

Junction-based lamellipodia drive endothelial cell rearrangements in vivo via a VE-cadherin/F-actin based oscillatory ratchet mechanism

Angiogenesis and vascular remodeling are driven by a wide range of endothelial cell behaviors, such as cell divisions, cell movements, cell shape and polarity changes. To decipher the cellular and molecular mechanism of cell movements, we have analyzed the dynamics of different junctional components during blood vessel anastomosis in vivo. We show that endothelial cell movements are associated with oscillating lamellipodia-like structures, which are orientated in the direction of these movements. These structures emerge from endothelial cell junctions and we thus call them junction-based lamellipodia (JBL). High-resolution time-lapse imaging shows that JBL are formed by F-actin based protrusions at the front end of moving cells. These protrusions also contain diffusely distributed VE-cadherin, whereas the junctional protein ZO-1 (Zona occludens 1) remains at the junction. Subsequently, a new junction is formed at the front of the JBL and the proximal junction is pulled towards the newly established distal junction. JBL function is highly dependent on F-actin dynamics. Inhibition of F-actin polymerization prevents JBL formation, whereas Rac-1 inhibition interferes with JBL oscillations. Both interventions disrupt endothelial junction formation and cell elongation. To examine the role of VE-cadherin (encoded by cdh5 gene) in this process, we generated a targeted mutation in VE-cadherin gene (cdh5ubs25), which prevents VE-cad/F-actin interaction. Although homozygous ve-cadherin mutants form JBL, these JBL are less dynamic and do not promote endothelial cell elongation. Taken together, our observations suggest a novel oscillating ratchet-like mechanism, which is used by endothelial cells to move along or over each other and thus provides the physical means for cell rearrangements.

developmental biology↗