Lymphatic endothelial-cell expressed ACKR3 is dispensable for postnatal lymphangiogenesis and lymphatic drainage function in mice

Atypical chemokine receptor ACKR3 (formerly CXCR7) is a scavenging receptor that has recently been implicated in murine lymphatic development. Specifically, ACKR3-deficiency was shown to result in lymphatic hyperplasia and lymphedema, in addition to cardiac hyperplasia and cardiac valve defects leading to embryonic lethality. The lymphatic phenotype was attributed to a lymphatic endothelial cell (LEC)-intrinsic scavenging function of ACKR3 for the vascular peptide hormone adrenomedullin (AM), which is also important during postnatal lymphangiogenesis. In this study, we investigated the expression of ACKR3 in the lymphatic vasculature of adult mice and its function in postnatal lymphatic development and function. We show that ACKR3 is widely expressed in mature lymphatics and that it exerts chemokine-scavenging activity in cultured murine skin-derived LECs. To investigate the role of LEC-expressed ACKR3 in postnatal lymphangiogenesis and function during adulthood, we generated and validated a lymphatic-specific, inducible ACKR3 knockout mouse. Surprisingly, in contrast to the reported involvement of ACKR3 in lymphatic development, our analyses revealed no contribution of LEC-expressed ACKR3 to postnatal lymphangiogenesis, lymphatic morphology and drainage function.

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Role of VEGF-D and VEGFR-3 in developmental lymphangiogenesis, a chemicogenetic study in Xenopus tadpoles

Blood ◽

10.1182/blood-2007-08-106302 ◽

2008 ◽

Vol 112 (5) ◽

pp. 1740-1749 ◽

Cited By ~ 31

Author(s):

Annelii Ny ◽

Marta Koch ◽

Wouter Vandevelde ◽

Martin Schneider ◽

Christian Fischer ◽

...

Keyword(s):

Critical Role ◽

Small Animal ◽

Lymphatic Endothelial Cell ◽

Lymph Vessel ◽

Small Animal Model ◽

Morpholino Knockdown ◽

Lymphatic Development ◽

Lymph Heart ◽

And Function

Abstract The importance of the lymphangiogenic factor VEGF-D and its receptor VEGFR-3 in early lymphatic development remains largely unresolved. We therefore investigated their role in Xenopus laevis tadpoles, a small animal model allowing chemicogenetic dissection of developmental lymphangiogenesis. Single morpholino antisense oligo knockdown of xVEGF-D did not affect lymphatic commitment, but transiently impaired lymphatic endothelial cell (LEC) migration. Notably, combined knockdown of xVEGF-D with xVEGF-C at suboptimal morpholino concentrations resulted in more severe migration defects and lymphedema formation than the corresponding single knockdowns. Knockdown of VEGFR-3 or treatment with the VEGFR-3 inhibitor MAZ51 similarly impaired lymph vessel formation and function and caused pronounced edema. VEGFR-3 silencing by morpholino knockdown, MAZ51 treatment, or xVEGF-C/D double knockdown also resulted in dilation and dysfunction of the lymph heart. These findings document a critical role of VEGFR-3 in embryonic lymphatic development and function, and reveal a previously unrecognized modifier role of VEGF-D in the regulation of embryonic lymphangiogenesis in frog embryos.

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Abstract 377: The Role of Impaired Lymphatic Drainage in Cardiac Transplantation

Circulation Research ◽

10.1161/res.127.suppl_1.377 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Sydney C Ginn ◽

Lanfang Wang ◽

Rebecca D Levit

Keyword(s):

Chronic Rejection ◽

Cardiac Transplantation ◽

Transplant Rejection ◽

Lymphatic Drainage ◽

Tissue Fluid ◽

Lymphatic Vasculature ◽

Lymphatic Development ◽

Cardiac Allografts ◽

Pro Inflammatory Cytokines

Functional lymphatic drainage inherently modulates cardiac function by maintaining the immune response and tissue-fluid homeostasis. During cardiac transplantation, the lymphatic collecting vessels are severed at the time of heart excision and not surgically reconstructed in the recipient. The consequence resulting from impaired lymphatic drainage in transplanted hearts is unknown. We hypothesize disruption of lymphatic drainage potentiates chronic inflammation by impeding the egress of immune cells and pro-inflammatory cytokines out of the myocardium exacerbating transplant rejection. Methods: Banked human allograft biopsies were utilized to retrospectively evaluate lymphatic differences between patients that did and did not develop chronic transplant rejection from 1 week to 5 years after surgery. Immunofluorescence staining permitted quantification of normalized lymphatic vessel number and area throughout the lifespan of each cardiac allograft (n=24). Autopsy patients with non-cardiac related fatalities served as controls to delineate normal cardiac lymphatic distribution (n=6). Results: Patients without chronic rejection displayed an initial presence of lymphatic vasculature that steadily declined (n=12), while patients with chronic rejection exhibited a delayed increase in lymphatic development (n=12). Conclusions: These data show significant differences in lymphatic area between patients with and without chronic transplant rejection at critical timepoints, suggesting delayed lymphangiogenesis may correlate with rejection. Translational Impact: These preliminary human data support further investigation into lymphatic-modifying therapeutics to prolong the life of cardiac allografts.

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Abstract 17171: Kruppel-Like Factor 4 Deletion Regulates Lymphatic Endothelial Cell Homeostasis

Circulation ◽

10.1161/circ.138.suppl_1.17171 ◽

2018 ◽

Vol 138 (Suppl_1) ◽

Author(s):

Victoria Mastej ◽

Cassondra Axen ◽

Kishore Wary

Keyword(s):

Endothelial Cell ◽

Fluorescent Protein ◽

Lymphatic Vessels ◽

Lymphatic Endothelial Cell ◽

Fluorescent Reporter ◽

Morphometric Analyses ◽

Bacterial Endotoxins ◽

Lymphatic Vasculature ◽

Green Fluorescent

Introduction and Hypothesis: Lymphatic vasculature exquisitely controls the clearance of metabolic end products, removal of bacterial endotoxins, and trafficking of immune cells. Signaling pathways that converge into endothelial cell kruppel-like factor 4 (KLF4) provide an atheroprotective role; however, the role of lymphatic endothelial cell (LEC)-KLF4 in these processes has not been clarified. Thus, we examined whether LEC-specific deletion of Klf4 alleles LECs. Methods and Results: After several generations of breeding, we transferred ROSA mT/mG (dual-fluorescent reporter allele mT/mG) to Prox1 CreERT2 (tamoxifen, TAM) inducible mice in C57 background to generate ROSA mT/mG ::Prox1 CreERT2 mice. In the next breeding scheme, ROSA mT/mG ::Prox1 CreERT2 female mice were crossed with male Klf4 fl/wt (Klf4 floxed allele) mice to generate heterozygous ROSA mT/mG ::Prox1 CreERT2 ::Klf4 fl/wt offspring (50%). Thereafter, we bred male ROSA mT/mG ::Prox1 CreERT2 ::Klf4 fl/wt with female ROSA mT/mG ::Prox1 CreERT2 ::Klf4 fl/wt to produce ROSA mT/mG ::Prox1 CreERT2 ::Klf4 fl/fl genotype (25%). These mice (12-14 weeks old) received TAM for five consecutive days, and on 21 st or 28 th day all mice were sacrificed. To confirm LEC Cre activation after TAM administration, we prepared cryosections of lung and the heart tissues. Mice receiving TAM showed lively and clear separation of green fluorescent protein (GFP) and tomato-Red colors, showing LEC specificity of Prox1 CreERT2 transgenic methodology. Cryosectioning combined with low- and high-resolution microscopy, and morphometric analyses of H&E sections showed disruption of LEC function and integrity in these mice. Biochemical experiments underway will address the role of KLF4 in lymphatic vessels homeostasis. Conclusion: These results indicate a key role for Klf4 in the regulation of lymphatic vessels.

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EphrinB2-EphB4 signalling provides Rho-mediated homeostatic control of lymphatic endothelial cell junction integrity

eLife ◽

10.7554/elife.57732 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Maike Frye ◽

Simon Stritt ◽

Henrik Ortsäter ◽

Magda Hernandez Vasquez ◽

Mika Kaakinen ◽

...

Keyword(s):

Endothelial Cell ◽

Barrier Function ◽

Lymphatic Vessel ◽

Lymphatic Vessels ◽

Lymphatic Endothelial Cell ◽

Cell Junction ◽

Adult Mice ◽

Junction Protein ◽

Vessel Integrity ◽

And Function

Endothelial integrity is vital for homeostasis and adjusted to tissue demands. Although fluid uptake by lymphatic capillaries is a critical attribute of the lymphatic vasculature, the barrier function of collecting lymphatic vessels is also important by ensuring efficient fluid drainage as well as lymph node delivery of antigens and immune cells. Here, we identified the transmembrane ligand EphrinB2 and its receptor EphB4 as critical homeostatic regulators of collecting lymphatic vessel integrity. Conditional gene deletion in mice revealed that EphrinB2/EphB4 signalling is dispensable for blood endothelial barrier function, but required for stabilization of lymphatic endothelial cell (LEC) junctions in different organs of juvenile and adult mice. Studies in primary human LECs further showed that basal EphrinB2/EphB4 signalling controls junctional localisation of the tight junction protein CLDN5 and junction stability via Rac1/Rho-mediated regulation of cytoskeletal contractility. EphrinB2/EphB4 signalling therefore provides a potential therapeutic target to selectively modulate lymphatic vessel permeability and function.

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Mechanosensation and Mechanotransduction by Lymphatic Endothelial Cells Act as Important Regulators of Lymphatic Development and Function

International Journal of Molecular Sciences ◽

10.3390/ijms22083955 ◽

2021 ◽

Vol 22 (8) ◽

pp. 3955

Author(s):

László Bálint ◽

Zoltán Jakus

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cell Fate ◽

Molecular Mechanisms ◽

Critical Role ◽

Mechanical Forces ◽

Lymphatic Endothelial Cells ◽

Lymphatic Development ◽

And Function ◽

The Impact

Our understanding of the function and development of the lymphatic system is expanding rapidly due to the identification of specific molecular markers and the availability of novel genetic approaches. In connection, it has been demonstrated that mechanical forces contribute to the endothelial cell fate commitment and play a critical role in influencing lymphatic endothelial cell shape and alignment by promoting sprouting, development, maturation of the lymphatic network, and coordinating lymphatic valve morphogenesis and the stabilization of lymphatic valves. However, the mechanosignaling and mechanotransduction pathways involved in these processes are poorly understood. Here, we provide an overview of the impact of mechanical forces on lymphatics and summarize the current understanding of the molecular mechanisms involved in the mechanosensation and mechanotransduction by lymphatic endothelial cells. We also discuss how these mechanosensitive pathways affect endothelial cell fate and regulate lymphatic development and function. A better understanding of these mechanisms may provide a deeper insight into the pathophysiology of various diseases associated with impaired lymphatic function, such as lymphedema and may eventually lead to the discovery of novel therapeutic targets for these conditions.

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Efficient aortic lymphatic drainage is necessary for atherosclerosis regression induced by ezetimibe

Science Advances ◽

10.1126/sciadv.abc2697 ◽

2020 ◽

Vol 6 (50) ◽

pp. eabc2697

Author(s):

Kim Pin Yeo ◽

Hwee Ying Lim ◽

Chung Hwee Thiam ◽

Syaza Hazwany Azhar ◽

Caris Tan ◽

...

Keyword(s):

Lymphatic Vessels ◽

Lymphatic Drainage ◽

Lymphatic Transport ◽

Atherosclerotic Plaques ◽

Tissue Fluid ◽

Atherosclerosis Progression ◽

Lymphatic Vasculature ◽

Lesion Regression ◽

Transport Of Macromolecules

A functional lymphatic vasculature is essential for tissue fluid homeostasis, immunity, and lipid clearance. Although atherosclerosis has been linked to adventitial lymphangiogenesis, the functionality of aortic lymphatic vessels draining the diseased aorta has never been assessed and the role of lymphatic drainage in atherogenesis is not well understood. We develop a method to measure aortic lymphatic transport of macromolecules and show that it is impaired during atherosclerosis progression, whereas it is ameliorated during lesion regression induced by ezetimibe. Disruption of aortic lymph flow by lymphatic ligation promotes adventitial inflammation and development of atherosclerotic plaque in hypercholesterolemic mice and inhibits ezetimibe-induced atherosclerosis regression. Thus, progression of atherosclerotic plaques may result not only from increased entry of atherogenic factors into the arterial wall but also from reduced lymphatic clearance of these factors as a result of aortic lymph stasis. Our findings suggest that promoting lymphatic drainage might be effective for treating atherosclerosis.

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The control of inflammation via the phosphorylation and dephosphorylation of tristetraprolin: a tale of two phosphatases

Biochemical Society Transactions ◽

10.1042/bst20160166 ◽

2016 ◽

Vol 44 (5) ◽

pp. 1321-1337 ◽

Cited By ~ 39

Author(s):

Andrew R. Clark ◽

Jonathan L.E. Dean

Keyword(s):

Inflammatory Mediators ◽

Knockout Mouse ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Human Orthologue ◽

And Function ◽

Detailed Picture ◽

Lines Of Evidence

Twenty years ago, the first description of a tristetraprolin (TTP) knockout mouse highlighted the fundamental role of TTP in the restraint of inflammation. Since then, work from several groups has generated a detailed picture of the expression and function of TTP. It is a sequence-specific RNA-binding protein that orchestrates the deadenylation and degradation of several mRNAs encoding inflammatory mediators. It is very extensively post-translationally modified, with more than 30 phosphorylations that are supported by at least two independent lines of evidence. The phosphorylation of two particular residues, serines 52 and 178 of mouse TTP (serines 60 and 186 of the human orthologue), has profound effects on the expression, function and localisation of TTP. Here, we discuss the control of TTP biology via its phosphorylation and dephosphorylation, with a particular focus on recent advances and on questions that remain unanswered.

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