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

The blood vasculature instructs lymphatic patterning in a SOX7-dependent manner

Despite a growing catalog of secreted factors critical for lymphatic network assembly, little is known about the mechanisms that modulate the expression level of these molecular cues in blood vascular endothelial cells (BECs). Here, we show that a BEC-specific transcription factor, SOX7, plays a crucial role in a non-cell-autonomous manner by modulating the transcription of angiocrine signals to pattern lymphatic vessels. While SOX7 is not expressed in lymphatic endothelial cells (LECs), the conditional loss of SOX7 function in mouse embryos causes a dysmorphic dermal lymphatic phenotype. We identify novel distant regulatory regions in mice and humans that contribute to directly repressing the transcription of a major lymphangiogenic growth factor (Vegfc) in a SOX7-dependent manner. Further, we show that SOX7 directly binds HEY1, a canonical repressor of the Notch pathway, suggesting that transcriptional repression may also be modulated by the recruitment of this protein partner at Vegfc genomic regulatory regions. Our work unveils a role for SOX7 in modulating downstream signaling events crucial for lymphatic patterning, at least in part via the transcriptional repression of VEGFC levels in the blood vascular endothelium.Peer reviewe

GATA2 is required for lymphatic vessel valve development and maintenance.

Heterozygous germline mutations in the zinc finger transcription factor GATA2 have recently been shown to underlie a range of clinical phenotypes, including Emberger syndrome, a disorder characterized by lymphedema and predisposition to myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). Despite well-defined roles in hematopoiesis, the functions of GATA2 in the lymphatic vasculature and the mechanisms by which GATA2 mutations result in lymphedema have not been characterized. Here, we have provided a molecular explanation for lymphedema predisposition in a subset of patients with germline GATA2 mutations. Specifically, we demonstrated that Emberger-associated GATA2 missense mutations result in complete loss of GATA2 function, with respect to the capacity to regulate the transcription of genes that are important for lymphatic vessel valve development. We identified a putative enhancer element upstream of the key lymphatic transcriptional regulator PROX1 that is bound by GATA2, and the transcription factors FOXC2 and NFATC1. Emberger GATA2 missense mutants had a profoundly reduced capacity to bind this element. Conditional Gata2 deletion in mice revealed that GATA2 is required for both development and maintenance of lymphovenous and lymphatic vessel valves. Together, our data unveil essential roles for GATA2 in the lymphatic vasculature and explain why a select catalogue of human GATA2 mutations results in lymphedema

In vitro assays using primary embryonic mouse lymphatic endothelial cells uncover key roles for FGFR1 signalling in lymphangiogenesis.

Despite the importance of blood vessels and lymphatic vessels during development and disease, the signalling pathways underpinning vessel construction remain poorly characterised. Primary mouse endothelial cells have traditionally proven difficult to culture and as a consequence, few assays have been developed to dissect gene function and signal transduction pathways in these cells ex vivo. Having established methodology for the purification, short-term culture and transfection of primary blood (BEC) and lymphatic (LEC) vascular endothelial cells isolated from embryonic mouse skin, we sought to optimise robust assays able to measure embryonic LEC proliferation, migration and three-dimensional tube forming ability in vitro. In the course of developing these assays using the pro-lymphangiogenic growth factors FGF2 and VEGF-C, we identified previously unrecognised roles for FGFR1 signalling in lymphangiogenesis. The small molecule FGF receptor tyrosine kinase inhibitor SU5402, but not inhibitors of VEGFR-2 (SU5416) or VEGFR-3 (MAZ51), inhibited FGF2 mediated LEC proliferation, demonstrating that FGF2 promotes proliferation directly via FGF receptors and independently of VEGF receptors in primary embryonic LEC. Further investigation revealed that FGFR1 was by far the predominant FGF receptor expressed by primary embryonic LEC and correspondingly, siRNA-mediated FGFR1 knockdown abrogated FGF2 mediated LEC proliferation. While FGF2 potently promoted LEC proliferation and migration, three dimensional tube formation assays revealed that VEGF-C primarily promoted LEC sprouting and elongation, illustrating that FGF2 and VEGF-C play distinct, cooperative roles in lymphatic vascular morphogenesis. These assays therefore provide useful tools able to dissect gene function in cellular events important for lymphangiogenesis and implicate FGFR1 as a key player in developmental lymphangiogenesis in vivo

Flame Front Analysis in Turbulent Combustion

aneous measurements of hydroxyl and formaldehyde laserinduced uorescence and total number densities byRayleigh scattering were carried out in a Bunsen type ame, which yields state of the art but noisy images. Nonlinear anisotropic diusion of edge-enhancing type [1] is shown here to enhance the capabilities of the laser-imaging technique in terms of ame front localization and correlation analysis. ######### Turbulentcombustion, ame front position, segmentation, anisotropic diusion ltering 1 Introduction Turbulent combustion processes are investigated in a close collaboration between experimental studies and numerical simulations in order to develop simulation tools that allow the optimization of technical combustion systems. In this interaction, ame front positions and structures as well as correlation between dierent scalars (species concentrations, temperatures) are essential to validate and further develop numerical simulation model

Down-Regulation of the miRNA-200 Family at the Invasive Front of Colorectal Cancers with Degraded Basement Membrane Indicates EMT Is Involved in Cancer Progression

Cancer progression is a complex series of events thought to incorporate the reversible developmental process of epithelial-to-mesenchymal transition (EMT). In vitro, the microRNA-200 family maintains the epithelial phenotype by posttranscriptionally inhibiting the E-cadherin repressors, ZEB1 and ZEB2. Here, we used in situ hybridization and immunohistochemistry to assess expression of miR-200 and EMT biomarkers in formalin-fixed paraffin-embedded human colorectal adenocarcinomas. In addition, laser capture microdissection and quantitative real-time polymerase chain reaction were employed to quantify levels of miR-200 in the normal epithelium, tumor core, invasive front, and stroma. We find that miR-200 is downregulated at the invasive front of colorectal adenocarcinomas that have destroyed and invaded beyond the basement membrane. However, regional lymph node metastases and vascular carcinoma deposits show strong expression of miR-200, suggesting this family of miRNAs is involved in the recapitulation of the primary tumor phenotype at metastatic sites. In contrast, adenomas and adenocarcinomas with intact basement membranes showed uniform miR-200 expression from the tumor core to the tumor-host interface. Taken together, these data support the involvement of EMT and mesenchymal-to-epithelial transition (MET) in the metastasis cascade and show that miR-200 is downregulated in the initial stages of stromal invasion but is restored at metastatic sites

FGF2 stimulates primary mouse LEC proliferation.

<p>(a) Primary LEC were cultured in EBM-2+0.5 mg ml⁻¹ Albumax (Control) or EBM-2+0.5 mg ml⁻¹ Albumax containing FGF2 or VEGFC at the indicated concentrations for 48 h. LEC proliferation was measured using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega). Data shown represent mean ± s.e.m. and are derived from 3 independent cell isolations, each prepared from multiple litters of embryos, and 5 replicates of each treatment (n = 15). (b) FGF2 stimulated LEC proliferation is inhibited by an FGFR tyrosine kinase inhibitor but not by VEGFR inhibitors. Primary LEC were cultured in EBM-2+0.5 mg ml⁻¹ Albumax (Control), or EBM-2+0.5 mg ml⁻¹ Albumax and FGF2 (10 ng ml⁻¹), together with the tyrosine kinase inhibitors SU5402 (10 µM, FGFR inhibitor), SU5416 (5 µM, VEGFR-2 inhibitor) or MAZ51 (5 µM, VEGFR-3 inhibitor). LEC proliferation was measured using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega). Data shown represent mean ± s.e.m. and are derived from 3 independent cell isolations prepared from multiple litters of embryos and 5 replicates of each treatment (n = 15). **<i>P</i><0.01 ***<i>P</i><0.001.</p

FGFR1 is important for LEC proliferation.

<p>(a) FGF receptor profile in primary embryonic mouse dermal LEC and BEC. Real-time RT-PCR analysis of <i>Fgfr1-4</i> mRNA levels in freshly isolated E16.5 LEC and BEC. Data are normalised to <i>Actb</i> and show mean ± s.d. of triplicate samples. Data are representative of at least three independent cell isolations from multiple litters of embryos. (b) FGFR1 is localized to the nucleus of LEC following stimulation with FGF2. Immunostaining of primary LEC cultured for 24 h in either EBM-2+0.5 mg ml⁻¹ Albumax (serum starved) or EGM-2MV containing FGF2 (complete media). Scale bars represent 40 µm. (c) siRNA mediated knockdown of FGFR1 in primary embryonic LEC. Primary LEC were cultured for 24 h prior to transfection with control or <i>Fgfr1</i> siRNA. <i>Fgfr1</i> mRNA levels were analysed 72 h post-transfection. Data are normalised to <i>Actb</i> and represent mean ± s.e.m. Data are derived from 3 independent cell isolations, each prepared from multiple litters of embryos, and 3 transfections per isolation (n = 9). ***<i>P</i><0.001. (d) FGFR1 protein levels were assessed by Western blot 72 h post-transfection and quantified relative to β-actin. (e) Immunostaining of primary LEC cultured in complete medium for 72 h after transfection with control or <i>Fgfr1</i> siRNA revealed efficient reduction in FGFR1 protein levels. Scale bars represent 100 µm. (f) FGFR1 is important for LEC proliferation. Primary LEC were cultured for 24 h prior to treatment with control or <i>Fgfr1</i> siRNA. LEC proliferation was measured by counting cells 72 h post-transfection. Data show mean ± s.e.m. Data are derived from 2 independent cell isolations, each prepared from multiple litters of embryos and multiple transfections per isolation (n = 11). ***<i>P</i><0.001. (g) Primary LEC were cultured in EGM-2MV (complete media, CM) for 24 h prior to treatment with SU5402 (10 µM) for 72 h. LEC proliferation was measured using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega). Data shown represent mean ± s.e.m. and are derived from 3 independent cell isolations prepared from multiple litters of embryos and multiple replicates of each treatment (n = 14). *<i>P</i><0.05.</p

FGF2 and VEGF-C promote tube formation of primary mouse LEC.

<p>(a) Primary LEC were cultured for 24 h and imaged immediately following the addition of Matrigel. (b) Primary LEC were cultured for 24 h followed by addition of Matrigel alone or Matrigel containing FGF2 (10 ng ml⁻¹), VEGF-C (200 ng ml⁻¹) or a combination of FGF2 and VEGF-C. Images were captured after a further 48 hours. (c) Primary LEC were cultured for 24 h followed by addition of Matrigel containing FGF2 (10 ng ml⁻¹) or a combination of FGF2 (10 ng ml⁻¹) and VEGF-C (200 ng ml⁻¹) and tyrosine kinase inhibitors SU5402 (10 µM, FGFR1), SU5416 (5 µM, VEGFR-2) or MAZ51 (5 µM, VEGFR-3). Three replicates of each treatment were performed and images are representative of at least three independent cell isolations. Inset panels in (c) illustrate magnified views of boxed regions. Scale bars represent 250 µm. Quantification of average vessel diameter (d) using Lymphatic Vessel Analysis Protocol (LVAP) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040497#pone.0040497-Shayan1" target="_blank">[28]</a> and ImageJ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040497#pone.0040497-Abramoff1" target="_blank">[29]</a> software and branch points per well (e) using AngioTool software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040497#pone.0040497-Zudaire1" target="_blank">[30]</a>, for each treatment indicated. Data show mean ± s.e.m. and are derived from 2 independent cell isolations, each prepared from multiple litters of embryos, and 3 replicates of each treatment (n = 6). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

FGF2 and VEGF-C promote migration of primary mouse LEC.

<p>(a) Confluent monolayers of primary LEC were scratched and cultured in EBM-2+0.5% FBS (Control), or EBM-2+0.5% FBS containing FGF2 (10 ng ml⁻¹) ± SU5402 (10 µM) or VEGF-C (200 ng ml⁻¹) for 8 h. Dotted white lines mark the boundaries of the wound at 0 h. Scale bars represent 125 µm. (b) Quantification of area migrated in 8 h. Data represent mean ± s.e.m. and are derived from 3 independent cell isolations, each prepared from multiple litters of embryos, and 5 replicates of each treatment (n = 15). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p