Myoferlin gene silencing decreases Tie-2 expression in vitro and angiogenesis in vivo Carol Yu, Arpeeta Sharma, Andy Trane, Soraya Utokaparch, Cleo Leung, Pascal Bernatchez ⁎ The Providence Heart and Lung Institute, The James Hogg Research Centre, St Paul's Hospital, University of British Columbia, Vancouver, British Columbia, Canada Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada abstract article info Article history: Received 17 October 2010 Received in revised form 13 April 2011 Accepted 18 April 2011 Keywords: Myoferlin Tie-2 Angiogenesis Angiogenesis consists in the growth of new blood vessels from pre-existing ones. Although anti-angiogenesis interventions have been shown to have therapeutic properties in human diseases such as cancer, their effect is only partial and the identification of novel modulators of angiogenesis is warranted. Recently, we reported the unexpected proteomic identification in endothelial cells (EC) of Myoferlin, a member of the Ferlin family of transmembrane proteins. Ferlins are well known to regulate the fusion of lipid vesicles at the plasma membrane in muscle cells, and we showed that Myoferlin gene knockdown not only decreases lipid vesicle fusion in EC but also attenuates Vascular Endothelial Growth Factor (VEGF) Receptor-2 (VEGFR-2) expression. Herein, we show that Myoferlin gene silencing in cultured EC also results in attenuated expression of a second tyrosine kinase receptor, Tie-2, which is another well-described angiogenic receptor. Most importantly, we provide evidence that delivery of a low-volume Myoferlin siRNA preparation in mouse tissues results in attenuated angiogenesis and edema formation. This provides the first evidence that acute Myoferlin knockdown has anti-angiogenic effects and validates Myoferlin as an anti-angiogenesis target. Furthermore, this supports the unexpected but increasingly accepted concept that proper tyrosine kinase receptors expression at the plasma membrane requires Myoferlin. © 2011 Elsevier Inc. All rights reserved. 1. Introduction Angiogenesis is well known to impact the outcome of a range of disorders, such as cancer and ischemic heart disease (Carmeliet, 2000). In settings of cancer, preventing angiogenesis decreases tumor size and favors patient survival in a synergistic fashion with chemotherapeutic agents (Folkman, 1975). The most common theory behind this synergy stipulates that anti-angiogenesis drugs not only prevent growth of new blood vessels, effectively starving tumors (Folkman, 1975), but also create a window of vessel stabilization which allows for better delivery of chemotherapeutic drugs (Hurwitz et al., 2004). However, the anti-tumor effect of angiogenesis blockers is partial and therefore the identification of novel angiogenesis-regulating pathways is essential. Through their tyrosine kinase receptors, members of the Vascular Endothelial Growth Factors (VEGFs) family of proteins have been described as the main endogenous regulators of angiogenesis. VEGFs' main isoform VEGF-A (VEGF) is up-regulated by ischemia, stimulates the proliferation and migration of endothelial cells (EC) (Ferrara, 2004), and in contrast to virtually any other growth factor, VEGF increases plasma protein permeability (Bernatchez et al., 1999, 2001; Sirois and Edelman, 1997). VEGF promotes angiogenesis in a synergistic fashion with other growth factors, such as angiopoietins (Ang), which are growth factors believed to regulate vascular quiescence. Ang-1 mediates its activity through its mostly EC-specific tyrosine kinase receptor Tie-2 (tyrosine kinase with Ig and epidermal growth factor homology domains-2), and downstream signaling results in greater vessel maturity and impermeability to vascular macromolecules (Jain and Munn, 2000). Hence, due to the importance of VEGFR-2 and Tie-2 for blood vessel growth, modulation of their surface expression is likely to have an effect on the outcome of angiogenesis-driven diseases, which is of therapeutic interest. Recently, by performing proteomics analysis of endothelial cholesterol-rich membrane microdomains (CEM), often referred to as caveolae/lipid rafts (Sharma et al., n.d.), we reported the unexpected detection in EC of muscle repair protein Myoferlin (Bernatchez et al., 2007, 2009), a member of the Ferlin protein family linked to muscular dystrophy (Bansal et al., 2003; Davis et al., 2000). Myoferlin exhibits a high degree of homology with the Caenorhabditis elegans FER-1 gene product that is essential for certain aspects of membrane fusion events at the plasma membrane (Argon and Ward, 1980; Ward et al., 1981; Achanzar and Ward, 1997). All Ferlins contain calcium-sensing and phospholipids-binding C2 domains that allow them to regulate membrane trafficking events at the plasmalemma (Glover and Brown, 2007; Washington and Ward, 2006). In EC, we found that Myoferlin gene silencing results in decreased EC proliferation, endocytosis and most importantly decreased translocation of VEGFR-2 to the plasma membrane resulting in its poly-ubiquitylation and proteasomal degradation (Bernatchez et al., 2007). This unexpectedly documented not only the presence of Ferlin in EC but also the fact that Vascular Pharmacology 55 (2011) 26–33 ⁎ Corresponding author at: The James Hogg Research Centre, St. Paul's Hospital, 1081 Burrard Street, Room 166, Vancouver, BC, Canada V6Z 1Y6. Tel.: +1 604 682 2344x66060; fax: +1 604 806 9274. E-mail address: pbernatc@interchange.ubc.ca (P. Bernatchez). 1537-1891/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.vph.2011.04.001 Contents lists available at ScienceDirect Vascular Pharmacology journal homepage: www.elsevier.com/locate/vph