Heterogeneity in VEGFR3 levels drives lymphatic vessel hyperplasia through cell-autonomous and non-cell-autonomous mechanisms

Incomplete delivery to the target cells is an obstacle for successful gene therapy approaches. Here we show unexpected effects of incomplete targeting, by demonstrating how heterogeneous inhibition of a growth promoting signaling pathway promotes tissue hyperplasia. We studied the function of the lymphangiogenic VEGFR3 receptor during embryonic and postnatal development. Inducible genetic deletion of Vegfr3 in lymphatic endothelial cells (LECs) leads to selection of non-targeted VEGFR3(+) cells at vessel tips, indicating an indispensable cell-autonomous function in migrating tip cells. Although Vegfr3 deletion results in lymphatic hypoplasia in mouse embryos, incomplete deletion during post-natal development instead causes excessive lymphangiogenesis. Analysis of mosaically targeted endothelium shows that VEGFR3(-) LECs non-cell-autonomously drive abnormal vessel anastomosis and hyperplasia by inducing proliferation of non-targeted VEGFR3(+) LECs through cell-contactdependent reduction of Notch signaling. Heterogeneity in VEGFR3 levels thus drives vessel hyperplasia, which has implications for the understanding of mechanisms of developmental and pathological tissue growth.Peer reviewe

Heparan sulfate expression in the neural crest is essential for mouse cardiogenesis

Impaired heparan sulfate (HS) synthesis in vertebrate development causes complex malformations due to the functional disruption of multiple HS-binding growth factors and morphogens. Here, we report developmental heart defects in mice bearing a targeted disruption of the HS-generating enzyme GlcNAc N-deacetylase/GlcN N-sulfotransferase 1 (NDST1), including ventricular septal defects (VSD), persistent truncus arteriosus (PTA), double outlet right ventricle (DORV), and retroesophageal right subclavian artery (RERSC). These defects closely resemble cardiac anomalies observed in mice made deficient in the cardiogenic regulator fibroblast growth factor 8 (FGF8). Consistent with this, we show that HS-dependent FGF8/FGF-receptor2C assembly and FGF8-dependent ERK-phosphorylation are strongly reduced in NDST1(-/-) embryonic cells and tissues. Moreover, WNT1-Cre/LoxP-mediated conditional targeting of NDST function in neural crest cells (NCCs) revealed that their impaired HS-dependent development contributes strongly to the observed cardiac defects. These findings raise the possibility that defects in HS biosynthesis may contribute to congenital heart defects in humans that represent the most common type of birth defect

Intrinsic coagulation pathway-mediated thrombin generation in mouse whole blood

Calibrated Automated Thrombography (CAT) is a versatile and sensitive method for analyzing coagulation reactions culminating in thrombin generation (TG). Here, we present a CAT method for analyzing TG in murine whole blood by adapting the CAT assay used for measuring TG in human plasma. The diagnostically used artificial and physiologic factor XII (FXII) contact activators kaolin, ellagic acid and polyphosphate (polyP) stimulated TG in murine blood in a dose-dependent manner resulting in a gradual increase in endogenous thrombin potential and peak thrombin, with shortened lag times and times to peak. The activated FXII inhibitor rHA-Infestin-4 and direct oral anticoagulants (DOACs) interfered with TG triggered by kaolin, ellagic acid and polyP and TG was completely attenuated in blood of FXII- (F12−/−) and FXI-deficient (F11−/−) mice. Moreover, reconstitution of blood from F12−/− mice with human FXII restored impaired contact-stimulated TG. HEK293 cell-purified polyP also initiated FXII-driven TG in mouse whole blood and addition of the selective inhibitor PPX_112 ablated natural polyP-stimulated TG. In conclusion, the data provide a method for analysis of contact activation-mediated TG in murine whole blood. As the FXII-driven intrinsic pathway of coagulation has emerged as novel target for antithrombotic agents that are validated in mouse thrombosis and bleeding models, our novel assay could expedite therapeutic drug development

Matrix stiffness controls lymphatic vessel formation through regulation of a GATA2-dependent transcriptional program

Tissue and vessel wall stiffening alters endothelial cell properties and contributes to vascular dysfunction. However, whether extracellular matrix (ECM) stiffness impacts vascular development is not known. Here we show that matrix stiffness controls lymphatic vascular morphogenesis. Atomic force microscopy measurements in mouse embryos reveal that venous lymphatic endothelial cell (LEC) progenitors experience a decrease in substrate stiffness upon migration out of the cardinal vein, which induces a GATA2-dependent transcriptional program required to form the first lymphatic vessels. Transcriptome analysis shows that LECs grown on a soft matrix exhibit increased GATA2 expression and a GATA2-dependent upregulation of genes involved in cell migration and lymphangiogenesis, including VEGFR3. Analyses of mouse models demonstrate a cell-autonomous function of GATA2 in regulating LEC responsiveness to VEGF-C and in controlling LEC migration and sprouting in vivo. Our study thus uncovers a mechanism by which ECM stiffness dictates the migratory behavior of LECs during early lymphatic development.Peer reviewe

Targeting NETs using dual-active DNase1 variants

Background: Neutrophil Extracellular Traps (NETs) are key mediators of immunothrombotic mechanisms and defective clearance of NETs from the circulation underlies an array of thrombotic, inflammatory, infectious, and autoimmune diseases. Efficient NET degradation depends on the combined activity of two distinct DNases, DNase1 and DNase1-like 3 (DNase1L3) that preferentially digest double-stranded DNA (dsDNA) and chromatin, respectively. Methods: Here, we engineered a dual-active DNase with combined DNase1 and DNase1L3 activities and characterized the enzyme for its NET degrading potential in vitro. Furthermore, we produced a mouse model with transgenic expression of the dual-active DNase and analyzed body fluids of these animals for DNase1 and DNase 1L3 activities. We systematically substituted 20 amino acid stretches in DNase1 that were not conserved among DNase1 and DNase1L3 with homologous DNase1L3 sequences. Results: We found that the ability of DNase1L3 to degrade chromatin is embedded into three discrete areas of the enzyme's core body, not the C-terminal domain as suggested by the state-of-the-art. Further, combined transfer of the aforementioned areas of DNase1L3 to DNase1 generated a dual-active DNase1 enzyme with additional chromatin degrading activity. The dual-active DNase1 mutant was superior to native DNase1 and DNase1L3 in degrading dsDNA and chromatin, respectively. Transgenic expression of the dual-active DNase1 mutant in hepatocytes of mice lacking endogenous DNases revealed that the engineered enzyme was stable in the circulation, released into serum and filtered to the bile but not into the urine. Conclusion: Therefore, the dual-active DNase1 mutant is a promising tool for neutralization of DNA and NETs with potential therapeutic applications for interference with thromboinflammatory disease states

Dissociation of VE-PTP from VE-cadherin is required for leukocyte extravasation and for VEGF-induced vascular permeability in vivo

Artificially stabilizing contacts between VE-cadherin and VE-PTP reduce vascular permeability and leukocyte extravasation in vivo

Expression profile of human lymphatic endothelial cells cultured on stiff (25 kPa) or soft (0.2 kPa) matrix conditions in the presence or absence of GATA2

Global transcriptome analysis showed that human lymphatic endothelial cells (LECs) grown on a soft matrix exhibit increased GATA2 expression, concomitant with a GATA2-dependent upregulation of genes involved in cell migration and lymphangiogenesis, including the key lymphangiogenic growth factor receptor VEGFR3. Affymetrix GeneChip analysis revealed regulation of 2771 transcripts above or below a 1.4-fold change (log2 fold change >0.5 or <-0.5) threshold on soft versus stiff matrices. Moreover, 406 (27%) of the 1485 transcripts that were increased and 207 (16 %) of the 1286 transcripts that were decreased on soft matrix were regulated in a GATA2 dependent manner. Overall design: In total 18 samples were analyzed (first biological replicate is uploaded here). To identify differentially expressed genes between the ctrl stiff and ctrl soft groups, a stepwise analysis with 6 biological replicates was performed. First, exon set ID’s with an average expression lower than 5 were considered as not significantly expressed and excluded from the analysis. A threshold of 40% increase (>0.5 log2 fold change) or decrease (0.5 log2 fold change) or decrease (<-0.5 log2 fold change) of gene expression in the absence of the GATA2 was used to generate the final list of genes

The importance of mechanical forces for in vitro endothelial cell biology

Blood and lymphatic vessels are lined by endothelial cells which constantly interact with their luminal and abluminal extracellular environments. These interactions confer physical forces on the endothelium, such as shear stress, stretch and stiffness, to mediate biological responses. These physical forces are often altered during disease, driving abnormal endothelial cell behavior and pathology. Therefore, it is critical that we understand the mechanisms by which endothelial cells respond to physical forces. Traditionally, endothelial cells in culture are grown in the absence of flow on stiff substrates such as plastic or glass. These cells are not subjected to the physical forces that endothelial cells endure , thus the results of these experiments often do not mimic those observed in the body. The field of vascular biology now realize that an intricate analysis of endothelial signaling mechanisms requires complex systems to mimic conditions. Here, we will review what is known about the mechanical forces that guide endothelial cell behavior and then discuss the advancements in endothelial cell culture models designed to better mimic the vascular microenvironment. A wider application of these technologies will provide more biologically relevant information from cultured cells which will be reproducible to conditions found in the body

Vegfr3-CreER (T2) mouse, a new genetic tool for targeting the lymphatic system

The lymphatic system is essential in many physiological and pathological processes. Still, much remains to be known about the molecular mechanisms that control its development and function and how to modulate them therapeutically. The study of these mechanisms will benefit from better controlled genetic mouse models targeting specifically lymphatic endothelial cells. Among the genes expressed predominantly in lymphatic endothelium, Vegfr3 was the first one identified and is still considered to be one of the best lymphatic markers and a key regulator of the lymphatic system. Here, we report the generation of a Vegfr3-CreER (T2) knockin mouse by gene targeting in embryonic stem cells. This mouse expresses the tamoxifen-inducible CreER(T2) recombinase under the endogenous transcriptional control of the Vegfr3 gene without altering its physiological expression or regulation. The Vegfr3-CreER (T2) allele drives efficient recombination of floxed sequences upon tamoxifen administration specifically in Vegfr3-expressing cells, both in vitro, in primary lymphatic endothelial cells, and in vivo, at different stages of mouse embryonic development and postnatal life. Thus, our Vegfr3-CreER (T2) mouse constitutes a new powerful genetic tool for lineage tracing analysis and for conditional gene manipulation in the lymphatic endothelium that will contribute to improve our current understanding of this system.Erratum in: Angiogenesis 19(3) p. 447 DOI: 10.1007/s10456-016-9518-5</p