Epsins are a family of ubiquitin-binding, endocytic clathrin adaptors. Mice lacking both epsins 1 and 2 (Epn1/2) die at embryonic day 10 and exhibit an abnormal vascular phenotype. To examine the angiogenic role of endothelial epsins, we generated mice with constitutive or inducible deletion of Epn1/2 in vascular endothelium. These mice exhibited no abnormal phenotypes under normal conditions, suggesting that lack of endothelial epsins 1 and 2 did not affect normal blood vessels. In tumors, however, loss of epsins 1 and 2 resulted in disorganized vasculature, significantly increased vascular permeability, and markedly retarded tumor growth. Mechanistically, we show that VEGF promoted binding of epsin to ubiquitinated VEGFR2. Loss of epsins 1 and 2 specifically impaired endocytosis and degradation of VEGFR2, which resulted in excessive VEGF signaling that compromised tumor vascular function by exacerbating nonproductive leaky angiogenesis. This suggests that tumor vasculature requires a balance in VEGF signaling to provide sufficient productive angiogenesis for tumor development and that endothelial epsins 1 and 2 negatively regulate the output of VEGF signaling. Promotion of excessive VEGF signaling within tumors via a block of epsin 1 and 2 function may represent a strategy to prevent normal angiogenesis in cancer patients who are resistant to anti-VEGF therapies.

SUMMARYNeuroligins are postsynaptic cell adhesion proteins that bind specifically to presynaptic membrane proteins called neurexins. Mutations in human neuroligin genes are associated with autism spectrum disorders in some families. The nematode Caenorhabditis elegans has a single neuroligin gene (nlg-1), and approximately a sixth of C. elegans neurons, including some sensory neurons, interneurons and a subset of cholinergic motor neurons, express a neuroligin transcriptional reporter. Neuroligin-deficient mutants of C. elegans are viable, and they do not appear deficient in any major motor functions. However, neuroligin mutants are defective in a subset of sensory behaviors and sensory processing, and are hypersensitive to oxidative stress and mercury compounds; the behavioral deficits are strikingly similar to traits frequently associated with autism spectrum disorders. Our results suggest a possible link between genetic defects in synapse formation or function, and sensitivity to environmental factors in the development of autism spectrum disorders. RESEARCH ARTICLENLG-1 is 26-28% identical (45-47% similar) to the four human neuroligins and is most similar overall (28% identical, 47% similar) to human neuroligin 4 (supplementary material Fig. S1).Through a combination of cDNA sequencing and reverse transcription PCR (RT-PCR) analysis of transcripts, we documented several types of nlg-1 alternative splicing (Figs 1, 2). Exons 13 and 14 are variably present in nlg-1 transcripts; the skipping of these two exons occurs independently, and we have detected transcripts containing only exon 13, only exon 14, both exons, and neither exon. In addition, we have identified tandem alternative splice acceptor sites at the 5Ј-ends of exons 4 and 16, and tandem alternative splice donor sites at the 3Ј-end of exon 14 (Figs 1, 2). If these splicing events are independent, there could be as many as 24 distinct NLG-1 isoforms. nlg-1 is expressed in a subset of neurons and muscle cellsWe used a transgenic transcriptional reporter, with the nlg-1 promoter driving YFP expression (FRM77, Fig. 1), to examine the cellular expression of nlg-1. We found that nlg-1 is expressed in a subset of neurons in C. elegans adults, including ~20 cells in the ventral nerve cord and ~20 cells in the head (Fig. 3). We identified the nlg-1-expressing cells in the ventral nerve cord as the cholinergic VA and DA motor neurons (Fig. 3). We also identified the two AIY and two URB interneurons and the four URA motor neurons in the head, and the two PVD mechanosensory and two HSN motor neurons in the body, as nlg-1-expressing cells. Of these cells, the AIY interneurons are cholinergic (Altun-Gultekin et al., 2001), the PVD neurons are glutamatergic (Lee et al., 1999), and the HSN neurons release both serotonin and acetylcholine (ACh) (Desai et al., 1988;Duerr et al., 2001). Neurotransmitter assignments have not been reported for the remaining nlg-1-expressing neurons; however, they do not express GABAergic, dopaminergic, serotonergic or glutamatergic repo...

Objective We previously showed that endothelial epsin deficiency causes elevated VEGFR2 and enhanced VEGF signaling, resulting in aberrant tumor angiogenesis and tumor growth in adult mice. However, direct evidence demonstrating that endothelial epsins regulate angiogenesis specifically through VEGFR2 downregulation is still lacking. In addition, whether the lack of epsins causes abnormal angiogenesis during embryonic development remains unclear. Approach and Results A novel strain of endothelial epsin-deleted mice that are heterozygous for VEGFR2 (Epn1fl/fl; Epn2−/−; Flkfl/+; iCDH5 Cre mice) was created. Analysis of embryos at different developmental stages shows that deletion of epsins causes defective embryonic angiogenesis and retards embryo development. In vitro angiogenesis assays using isolated primary endothelial cells (EC) from Epn1fl/fl; Epn2−/−; iCDH5 Cre (EC-iDKO) and Epn1fl/fl; Epn2−/−; Flkfl/+; iCDH5 Cre (EC-iDKO-Flkfl/+) mice demonstrated that VEGFR2 reduction in epsin depleted cells is sufficient to restore normal VEGF signaling, EC proliferation, EC migration and EC network formation. These findings were complemented by in vivo wound healing, inflammatory angiogenesis, and tumor angiogenesis assays in which reduction of VEGFR2 was sufficient to rescue abnormal angiogenesis in endothelial epsin-deleted mice. Conclusions Our results provide the first genetic demonstration that epsins function specifically to downregulate VEGFR2 by mediating activated VEGFR2 internalization and degradation and that genetic reduction of VEGFR2 level protects against excessive angiogenesis caused by epsin loss. Our findings indicate epsins may be a potential therapeutic target in conditions where tightly regulated angiogenesis is crucial, such as in diabetic wound healing and tumors.