Genome-wide functional analysis reveals central signaling regulators of lymphatic endothelial cell migration and remodeling

Abstract

Lymphatic vessels constitute a specialized vasculature that is involved in development, cancer, obesity, and immune regulation. The migration of lymphatic endothelial cells (LECs) is critical for vessel growth (lymphangiogenesis) and vessel remodeling, processes that modify the lymphatic network in response to developmental or pathological demands. Using the publicly accessible results of our genome-wide siRNA screen, we characterized the migratome of primary human LECs and identified individual genes and signaling pathways that regulate LEC migration. We compared our data set with mRNA differential expression data from endothelial and stromal cells derived from two in vivo models of lymphatic vessel remodeling, viral infection and contact hypersensitivity-induced inflammation, which identified genes selectively involved in regulating LEC migration and remodeling. We also characterized the top candidates in the LEC migratome in primary blood vascular endothelial cells to identify genes with functions common to lymphatic and blood vascular endothelium. On the basis of these analyses, we showed that , which encodes the glycan-binding protein Galectin-1, promoted lymphatic vascular growth in vitro and in vivo and contributed to maintenance of the lymphatic endothelial phenotype. Our results provide insight into the signaling networks that control lymphangiogenesis and lymphatic remodeling and potentially identify therapeutic targets and biomarkers in disease specific to lymphatic or blood vessels.

Consensus guidelines for the use and interpretation of angiogenesis assays

“…Large genome-wide screens can also be assessed with the scratch wounding technique. The use of precision wounding replicators with floating pins and a workstation robot enables large numbers of scratches to be made with reduced coefficients of variation [ 43 , 44 ].…”

“…Electric cell-substrate impedance sensing (ECIS, Applied Biophysics Inc, New York, USA) can also be used to automatically create wounds and simultaneously measure cell migration in multiple wells, and V&P Scientific has developed a wounding replicator using floating pins that deliver a precise scratch ( www.vp-scientific.com/wounding_tissue_culture_experiments.php ) by a Sciclone ALH 3000 workstation robot (Caliper Life Sciences, Hopkinton, MA). The additional precision of the scratches using the robot over manual wounding replicators makes the latter instrument the method of choice when performing the scratch wound assay for large-scale screens on drug or genome-wide siRNA/sgRNA screens [ 43 , 44 ].…”

Galectins: Multitask signaling molecules linking fibroblast, endothelial and immune cell programs in the tumor microenvironment

“…Notably, Gal-1 blockade also induced vessel normalization as revealed by pericyte coverage of ECs, reduction of tumor hypoxia and augmented influx of immune cells into the tumor parenchyma [81], suggesting a major role for this lectin in connecting ECs, pericytes and immune cell networks. Interestingly, genome-wide functional analysis recently identified Gal-1 as a pivotal regulator of lymphatic vascular growth and maintenance of the lymphatic endothelial phenotype via lectin interaction with VEGFR2 [87].…”

Emerging Roles for VEGF-D in Human Disease

“…Importantly, insight into the function of this protein has led to improvements in the diagnosis of the devastating pulmonary disease lymphangioleiomyomatosis (LAM) [ 3 , 4 ] so research into the structure/function relationship, biochemistry and biology of VEGF-D in cell-based systems, animal models and clinicopathological studies has already led to improved clinical practice. Opportunities for further clinical impact are being developed through systems biology approaches to build a comprehensive understanding of the molecular signaling networks controlling the remodeling of blood vessels and lymphatics in disease [ 5 , 6 ], thereby enabling us to delineate how VEGF-D signaling is integrated with other signaling systems in the endothelial cells of blood vessels and lymphatics. These signaling systems are central to key biological processes in vascular biology such as cell proliferation, migration, tube formation and regression of excess vessels.…”