Biology subjects

Claesson-Welsh, L.

Publications and source records attributed to Claesson-Welsh, L..

11 recordsLinked to original sources

bioRxiv2025

Lymphatics unload VEGFR3 in response to the endothelial stress upon VEGFR2 insufficiency

Vascular endothelial growth factor receptor 2 (VEGFR2) is a key target for regulating the endothelial cell lineage and angiogenesis. It is also expressed by lymphatic endothelial cells (LEC) while its participation in the process of lymphangiogenesis remains inadequately characterized. We show in this study that VEGFR2 is highly expressed in dermal initial lymphatic vessels and valves. The induced deletion of pan-endothelial Vegfr2 at the neonatal stage produced a potent suppression of dermal lymphatic growth, including the thinner lymphatic diameter, the decrease of LEC number and lymphatic valves. Mechanistically, the VEGFR2 insufficiency led to a dramatic decrease of lymphatic VEGFR3, a key regulator mediating signals for lymphatic growth and remodeling. The RNA sequencing analysis revealed that GO terms enriched for the downregulated genes included biological processes related to the EC development while pathways related to the hematopoiesis and immune responses were upregulated in the skin of Vegfr2 mutants compared with the littermate controls. This was further confirmed by the RNA-seq analysis of dermal tissues 48 hours after the endothelial Vegfr2 deletion. Consistently, an inhibitory effect on the dermal lymphatic growth was also observed by targeting Vegfr2 in PROX1+ cells, manifesting a similarly altered transcriptome signature. The alteration of lymphatic gene expression was further validated by the siRNA mediated knockdown of Vegfr2 in primary lymphatic endothelial cells, showing a transcriptional trend of LEC to hematopoietic transition. Findings from this study imply that VEGFR2 is required for the maintenance of endothelial identity and its insufficiency may trigger the alteration of lymphatic transcriptional programs to diminish the VEGFR3-mediated lymphangiogenesis.

developmental biology↗

bioRxiv2024

Neuropilin-1 controls vascular permeability through juxtacrine regulation of endothelial adherens junctions

Neuropilin-1 (NRP1) regulates endothelial cell (EC) biology through modulation of vascular endothelial growth factor receptor 2 (VEGFR2) signalling by presenting VEGFA to VEGFR2. How NRP1 impacts VEGFA-mediated vascular hyperpermeability has however remained unresolved, described as exerting either a positive or a passive function. Using EC-specific Nrp1 knock-out mice, we discover that EC-expressed NRP1 exerts an organotypic role. In the ear skin, VEGFA/VEGFR2-mediated vascular leakage was increased following loss of EC NRP1, implicating NRP1 in negative regulation of VEGFR2 signalling. In contrast, in the back skin and trachea, loss of EC NRP1 decreased vascular leakage. In accordance, phosphorylation of vascular endothelial (VE)-cadherin was increased in the ear skin but suppressed in the back skin of Nrp1 iECKO mice. NRP1 expressed on perivascular cells has been shown to impact VEGF-mediated VEGFR2 signalling. Importantly, expression of NRP1 on perivascular cells was more abundant in the ear skin than in the back skin. Global loss of NRP1 resulted in suppressed VEGFA-induced vascular leakage in the ear skin, implicating perivascular NRP1 as a juxtacrine co-receptor of VEGFA in this compartment. Altogether, we demonstrate that perivascular NRP1 is an active participant in EC VEGFA/VEGFR2 signalling and acts as an organotypic modifier of EC biology.

cell biology↗

bioRxiv2023

A critical role of VEGFR2 in lymphatic tumor metastasis

Vascular endothelial growth factor receptor-2 (VEGFR2) transduces crucial signals for blood vessel growth but its role in the lymphatic system remains incompletely elucidated. By employing genetic mouse models targeting Vegfr2 in either pan-endothelial cells (ECs) or lymphatic endothelial cells (LECs), we examined roles of VEGFR2 in lymphangiogenesis and in tumor progression. VEGFR2 was differentially expressed in the murine lymphatic system and particularly marked in valves of collecting vessels. The pan-endothelial Vegfr2 deletion (Vegfr2iECKO) reduced the dermal lymphatic growth, and a significant decrease in lymphatic valves of pre-collectors was observed in mice with the LEC-specific attenuation of VEGFR2 (Vegfr2iLECKO). Furthermore, while the primary growth of subcutaneously implanted Lewis lung carcinoma was unaffected in the Vegfr2iLECKO mouse model, the tumor metastasis to sentinel lymph nodes was efficiently suppressed. In accordance, the tumor-associated lymphangiogenesis was decreased in the Vegfr2iLECKO mice compared with the control. Findings from this study imply that the lymphatic VEGFR2 regulates valve morphogenesis and promotes lymph node metastasis by regulating the tumor-associated lymphatic formation.

cancer biology↗

bioRxiv2022

Claudin5 protects the peripheral endothelial barrier in an organ and vessel type-specific manner

The pathogenesis of numerous diseases is characterised by disruption of the junctions that form the endothelial cell (EC) barrier, the composition of which may differ greatly between organs. However, the expression level variability and precise contribution of different junctional proteins is poorly understood. Here, we focus on organs with continuous endothelium to identify structural and functional in vivo characteristics of the EC barrier. Assembly of multiple single-cell RNAseq datasets into a single integrated database revealed the variability in EC barrier patterning. Across tissues Claudin5 exhibited diminishing expression along the arteriovenous axis, which correlates with EC barrier integrity. Functional analysis identified tissue-specific differences in leakage patterning and response to agonist-induced leakage. We uncover that Claudin5 loss enhances agonist-induced leakage in an organotypic, vessel type-specific and size-selective manner in an inducible, EC-specific, knock-out mouse. Mechanistically, Claudin5 loss induces no change in junction ultrastructure but alters composition, with concomitant loss of zonula occludens-1 (ZO-1) expression and upregulation of VE-Cadherin. These findings uncover the organ-specific organisation of the EC barrier and distinct importance of Claudin5 in different vascular beds and will aid our ability to modify EC barrier stability in a targeted, organ-specific manner.

cell biology↗

bioRxiv2022

p73 controls cell junction dynamics during sprouting angiogenesis and acts via Angiomotin

Preservation of blood vessels integrity, which is critical for normal physiology and organ function, is controlled at multiple levels, including endothelial junctions. However, the mechanism that controls the adequate assembly of endothelial cell junctions is not fully defined. Here we uncover TAp73 transcription factor as a vascular architect that orchestrates transcriptional programs involved in cell junction establishment and developmental blood vessel morphogenesis and identify Angiomotin (AMOT) as a TAp73 direct transcriptional target. Knockdown of p73 in endothelial cells not only results in decreased Angiomotin expression and localization at intercellular junctions, but also affects its downstream function regarding Yes-Associated Protein (YAP) cytoplasmic sequestration upon cell-cell contact. Analysis of adherens junctional morphology after p73-knockdown in human endothelial cells revealed striking alterations, particularly a sharp increase in serrated junctions and actin bundles appearing as stress fibers, both features associated with enhanced barrier permeability. In turn, stabilization of Angiomotin levels rescued those junctional defects, confirming that TAp73 controls endothelial junction dynamics, at least in part, through the regulation of Angiomotin. The observed defects in monolayer integrity were linked to hyperpermeability and reduced transendothelial electric resistance. Moreover, p73-knockout retinas showed a defective sprout morphology coupled to hemorrhages, highlighting the physiological relevance of p73 regulation in the maintenance of vessel integrity in vivo. We propose a new model in which TAp73 acts as a vascular architect integrating transcriptional programs that will impinge with Angiomotin/YAP signaling to maintain junctional dynamics and integrity, whilst balancing endothelial cell rearrangements in angiogenic vessels.

cell biology↗

bioRxiv2021

Endothelial Unc5B controls blood-brain barrier integrity

Blood-brain barrier (BBB) integrity is critical for proper function of the central nervous system (CNS). Here, we showed that the endothelial Netrin1 receptor Unc5B controls BBB integrity by maintaining Wnt/{beta}-catenin signaling. Inducible endothelial-specific deletion of Unc5B in adult mice led to region and size-selective BBB opening. Loss of Unc5B decreased BBB Wnt/{beta}-catenin signaling, and {beta}-catenin overexpression rescued Unc5B mutant BBB defects. Mechanistically, Netrin1 enhanced Unc5B interaction with the Wnt co-receptor LRP6, induced its phosphorylation and activated Wnt/{beta}-catenin downstream signaling. Intravenous delivery of antibodies blocking Netrin1 binding to Unc5B caused a transient disruption of Wnt signaling and BBB breakdown, followed by neurovascular barrier resealing. These data identify Netrin-Unc5B signaling as a novel regulator of BBB integrity with potential therapeutic utility for CNS diseases.

cell biology↗

bioRxiv2020

eNOS-induced vascular barrier disruption in retinopathy by c-Src activation and tyrosine phosphorylation of VE-cadherin

Hypoxia and the production of vascular endothelial growth factor A (VEGFA) promote blood vessel leakiness and edema in ocular diseases. Therapeutics targeting VEGFA suppress leakiness and edema but aggravate hypoxia; therefore, new therapeutics are needed. We examined the role of endothelial nitric oxide synthase (eNOS) in pathological neovascularization and vessel permeability during oxygen-induced retinopathy. NO formation was suppressed chemically using L-NMMA, or genetically, in eNOS serine to alanine (S1176A) mutant mice, resulting in reduced retinal neoangiogenesis. Both strategies resulted in reduced vascular leakage by stabilizing endothelial adherens junctions through suppressed phosphorylation of vascular endothelial (VE)-cadherin Y685 in a c-Src-dependent manner. Intervention treatment by a single dose of L-NMMA in established retinopathy restored the vascular barrier and prevented leakage. We conclude that eNOS induces destabilization of adherens junctions and vascular hyperpermeability by converging with the VEGFA/VEGFR2/c-Src/VE-cadherin pathway and that this pathway can be selectively inhibited by blocking NO formation.

cell biology↗

bioRxiv2020

Pre-determined macromolecular leakage sites with dynamic properties establish endothelial intra-vessel heterogeneity

Endothelial cells display heterogeneous properties based on location and function. How this heterogeneity influences endothelial barrier stability both between and within vessel subtypes is unexplored. We find here that endothelial cells exhibit heterogeneous barrier properties on inter-organ and intra-vessel levels. Using intravital microscopy and sequential stimulation of the ear dermis with vascular endothelial growth factor-A and/or histamine, we observe distinct, reappearing sites, common for both agonists, where leakage preferentially takes place. Further, through repetitive stimulation of the diaphragm and trachea, we find inter-organ conservation of such leakage sites. Qualitatively, pre-determined sites display distinct leakage properties and enhanced barrier breakdown compared to less susceptible regions. Mechanistically, pre-determined sites exhibit lower laminin 5 deposition, which correlates with reduced junctional vascular endothelial (VE)-Cadherin. These data thus highlight functional intra-vessel heterogeneity that defines pre-determined sites which display distinct leakage properties and which may have great impact on pathological vascular leakage and drug delivery.

cell biology↗

bioRxiv2020

Paladin is a PI(4,5)P2 phosphoinositide phosphatase that regulates endosomal signaling and angiogenesis

Cell signaling governs cellular behavior and is therefore subject to tight spatiotemporal regulation. Signaling output is regulated by specialized cell membranes and vesicles which contain unique combinations of lipids and proteins. The phospholipid phosphatidylinositol 4,5-bisphosphate, (PI(4,5)P2), an important component of the plasma membrane as well as other subcellular membranes, is involved in multiple processes, including signaling. However, which enzymes drive the formation and degradation of non-plasma membrane PI(4,5)P2, and their impact on cell signaling and function at the organismal level are unknown. Here we show in a mouse model that Paladin is a vascular PI(4,5)P2 phosphatase that regulates endosomal signaling and angiogenesis. Paladin was localized to the endosomal and Golgi compartments, and interacted with vascular endothelial growth factor receptor 2 (VEGFR2) in vitro and in vivo. Loss of Paladin resulted in increased internalization of the receptor, over-activation of extracellular regulated kinase, and hypersprouting of endothelial cells in the developing retina of mice. These findings suggest that inhibition of Paladin, or other endosomal PI(4,5)P2 phosphatases, could be exploited to modulate VEGFR2 signaling and angiogenesis, when direct and full inhibition of the receptor is not desirable.

cell biology↗

bioRxiv2019

Caspase-8 Modulates Angiogenesis By Regulating A Cell Death Independent Pathway In Endothelial Cells

During developmental angiogenesis blood vessels grow and remodel to ultimately build a hierarchical vascular network. Whether and how cell death signaling molecules contribute to blood vessel formation is still not well understood. Caspase-8 (Casp-8), a key protease in the extrinsic cell death-signaling pathway, regulates both cell death via apoptosis and necroptosis. Here we show that expression of Casp-8 in endothelial cells (ECs) is required for proper postnatal angiogenesis. EC specific Casp-8 knockout pups (Casp-8ECko) have reduced retinal angiogenesis, as the loss of Casp-8 reduced EC proliferation, sprouting and migration independent of its cell death function. Instead, the loss of Casp-8 caused hyperactivation of p38 mitogen-activated protein kinase (MAPK) downstream of receptorinteracting serine/threonine-protein kinase 3 (RIPK3) and destabilization of VE-cadherin at EC junctions. In a mouse model of oxygen-induced retinopathy (OIR), resembling retinopathy of prematurity (ROP), loss of Casp-8 in ECs is beneficial, as pathological neovascularization was reduced in Casp-8ECko pups. Taken together, we identify that Casp-8 signals in a cell-death independent manner in ECs during postnatal and pathological blood vessel formation.

developmental biology↗

bioRxiv2019

Mouse retinal cell behaviour in space and time using light sheet fluorescence microscopy

As the general population ages and the incidence of diabetes increases epidemically, more people are affected by eye diseases, such as retinopathies. It is therefore critical to improve imaging of eye disease mouse models. Here, we demonstrate that 1) rapid, quantitative 3D and 4D (time lapse) imaging of cellular and subcellular processes in the murine eye is feasible, with and without tissue clearing, using light-sheet fluorescent microscopy (LSFM) and 2) LSFM readily reveals new features of even well studied eye disease mouse models, such as the Oxygen-Induced Retinopathy (OIR) model. Through correlative LSFM-Confocal imaging we find that flat-mounting retinas for confocal microscopy significantly distorts tissue morphology. The minimized distortion with LSFM dramatically improved analysis of pathological vascular tufts in the OIR model revealing \"knotted\" morphologies, leading to a proposed new tuft nomenclature. Furthermore, live-imaging of OIR tuft formation revealed abnormal cell motility and altered filopodia dynamics. We conclude that quantitative 3D/4D LSFM imaging and analysis has the potential to advance our understanding of pathological processes in the eye, in particular neuro-vascular degenerative processes.

developmental biology↗