Abstract
Vascular endothelial growth factors (VEGFs) are key mediators of endothelial cell (EC) function in angiogenesis. Emerging knowledge also supports the involvement of axon guidance-related factors in the regulation of angiogenesis and vascular patterning. In the current study, we demonstrate that fibronectin and leucine-rich transmembrane protein-3 (FLRT3), an axon guidance-related factor connected to the regulation of neuronal cell outgrowth and morphogenesis but not to VEGF-signaling, was upregulated in ECs after VEGF binding to VEGFR2. We found that FLRT3 exhibited a transcriptionally paused phenotype in non-stimulated human umbilical vein ECs. After VEGF-stimulation its nascent RNA and mRNA-levels were rapidly upregulated suggesting that the regulation of FLRT3 expression is mainly occurring at the level of transcriptional elongation. Blockage of FLRT3 by siRNA decreased survival of ECs and their arrangement into capillary-like structures but enhanced cell migration and wound closure in wound healing assay. Bifunctional role of FLRT3 in repulsive vs. adhesive cell signaling has been already detected during embryogenesis and neuronal growth, and depends on its interactions either with UNC5B or another FLRT3 expressed by adjacent cells. In conclusion, our findings demonstrate that besides regulating neuronal cell outgrowth and morphogenesis, FLRT3 has a novel role in ECs via regulating VEGF-stimulated EC-survival, migration, and tube formation. Thus, FLRT3 becomes a new member of the axon guidance-related factors which participate in the VEGF-signaling and regulation of the EC functions.
Highlights
Angiogenesis is indispensable for development, growth and regeneration of all tissues by supplying nutrients and oxygen (Persson and Buschmann, 2011)
A role of fibronectin and leucine-rich transmembrane protein3 (FLRT3) in the regulation of endothelial cell (EC) function remains ill-defined and there is no previous data about its connection to Vascular endothelial growth factors (VEGFs)-signaling
We show for the first time that FLRT3: (1) exhibits a paused phenotype in non-stimulated HUVECs, (2) is rapidly upregulated in ECs after stimulation of VEGFR-2 with VEGFs; and (3) has a role in the regulation of VEGF-induced survival and migration of ECs as well as in vitro angiogenesis
Summary
Angiogenesis is indispensable for development, growth and regeneration of all tissues by supplying nutrients and oxygen (Persson and Buschmann, 2011). Sprouting angiogenesis is initiated when endothelial cells (ECs) in the vessel wall, called tip cells, are polarized leading to their extension and migration toward an angiogenic stimulus. Other ECs in the vessel wall, called stalk cells, proliferate and form a cord for the developing sprout of the vessel. VEGF-Induced FLRT3 Expression in ECs of the sprout continues until a tip cell reaches another tip cell or a small vessel and forms a new connection (Risau, 1997; Ribatti and Crivellato, 2012). Lumen formation is there after facilitated by intracellular vacuolization or cell–cell repulsion, i.e., by local repulsion of cell surfaces of the neighboring ECs in the newly formed non-lumenized cord (Kamei et al, 2006; Strilicet al., 2010). Vessel maturation is promoted by recruitment of pericytes as well as assembly and production of basement membrane (Stratman et al, 2009; Davis et al, 2015)
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