The formin FMNL3 is a cytoskeletal regulator of angiogenesis.

The process of angiogenesis requires endothelial cells (ECs) to undergo profound changes in shape and polarity. Although this must involve remodelling of the EC cytoskeleton, little is known about this process or the proteins that control it. We used a co-culture assay of angiogenesis to examine the cytoskeleton of ECs actively undergoing angiogenic morphogenesis. We found that elongation of ECs during angiogenesis is accompanied by stabilisation of microtubules and their alignment into parallel arrays directed at the growing tip. In other systems, similar microtubule alignments are mediated by the formin family of cytoskeletal regulators. We screened a library of human formins and indentified formin-like 3 (FMNL3; also known as FRL2) as a crucial regulator of EC elongation during angiogenesis. We showed that activated FMNL3 triggers microtubule alignment and that FMNL3 is required for this alignment during angiogenic morphogenesis. FMNL3 was highly expressed in the ECs of zebrafish during development and embryos that were depleted for FMNL3 showed profound defects in developmental angiogenesis that were rescued by expression of the human gene. We conclude that FMNL3 is a new regulator of endothelial microtubules during angiogenesis and is required for the conversion of quiescent ECs into their elongated angiogenic forms.

Uses of the in vitro endothelial-fibroblast organotypic co-culture assay in angiogenesis research.

Angiogenesis is a complex process that involves multiple cellular events. In addition to receiving inputs from a range of stimulatory and inhibitory factors, endothelial cells undergoing angiogenesis make multiple interactions with the extracellular matrix and with other cell types in the stroma. Recreating angiogenesis in vitro is probably an impossible goal; however, a number of assays have been developed that recapitulate many of the key events of the process. These assays are indispensible tools for investigating the signalling pathways that control the formation of new blood vessels. In the present paper, we review the organotypic co-culture assay of angiogenesis - until recently, a comparatively underemployed assay, but one with a number of powerful advantages for angiogenesis research. We give a set of optimized protocols for its use, including protocols for siRNA (small interfering RNA)-based screens, and we discuss appropriate methods for obtaining quantitative data from the assay.

Farnesyltransferase inhibitors target multiple endothelial cell functions in angiogenesis.

Farnesyltransferase inhibitors (FTIs) are novel anticancer drugs that inhibit the secretion of pro-angiogenic factors by Ras-transformed cancer cells. FTIs also inhibit angiogenesis in a rat corneal model, suggesting that FTIs have anti-angiogenic properties that extend beyond targeting cancer cells. Our hypothesis was that FTIs may directly target endothelial cell functions in angiogenesis. We examined the effects of FTI treatment on a range of assays designed to pick apart the individual functions of endothelial cells during angiogenesis. We found that FTIs inhibit endothelial cell proliferation, causing a failure of mitosis and accumulation of binucleate cells. FTIs also block the directional migration of endothelial cells toward VEGF, the major pro-angiogenic factor in adult tissues. In a co-culture assay of angiogenesis, FTI treatment significantly inhibits tube formation, but has no effect on pre-existing structures. Defects in tube formation could be replicated by specific targeting of endothelial cell farnesyltransferase using RNA interference. Our data show that FTIs directly target endothelial cells in angiogenesis, explaining previous in vivo findings. Importantly, these results suggest that the therapeutic use of FTIs may extend beyond cancer to include the treatment of other diseases involving pathological angiogenesis.

The Formin FMNL3 Controls Early Apical Specification in Endothelial Cells by Regulating the Polarized Trafficking of Podocalyxin.

Angiogenesis is the fundamental process by which new blood vessels form from pre-existing vasculature. It plays a critical role in the formation of the vasculature during development and is triggered in response to tissue hypoxia in adult organisms. This process requires complex and coordinated regulation of the endothelial cell cytoskeleton to control cell shape and polarity. In our previous work, we showed that the cytoskeletal regulator FMNL3/FRL2 controls the alignment of stabilized microtubules during polarized endothelial cell elongation and that depletion of FMNL3 retards elongation of the intersegmental vessels in zebrafish. Recent work has shown that FMNL3 is also needed for vascular lumen formation, a critical element of the formation of functional vessels. Here, we show that FMNL3 interacts with Cdc42 and RhoJ, two Rho family GTPases known to be required for lumen formation. FMNL3 and RhoJ are concentrated at the early apical surface, or AMIS, and regulate the formation of radiating actin cables from this site. In diverse biological systems, formins mediate polarized trafficking through the generation of similar actin filaments tracks. We show that FMNL3 and RhoJ are required for polarized trafficking of podocalyxin to the early apical surface--an important event in vascular lumenogenesis.