miR-126 regulates angiogenic signaling and vascular integrity.

Precise regulation of the formation, maintenance, and remodeling of the vasculature is required for normal development, tissue response to injury, and tumor progression. How specific microRNAs intersect with and modulate angiogenic signaling cascades is unknown. Here, we identified microRNAs that were enriched in endothelial cells derived from mouse embryonic stem (ES) cells and in developing mouse embryos. We found that miR-126 regulated the response of endothelial cells to VEGF. Additionally, knockdown of miR-126 in zebrafish resulted in loss of vascular integrity and hemorrhage during embryonic development. miR-126 functioned in part by directly repressing negative regulators of the VEGF pathway, including the Sprouty-related protein SPRED1 and phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2/p85-beta). Increased expression of Spred1 or inhibition of VEGF signaling in zebrafish resulted in defects similar to miR-126 knockdown. These findings illustrate that a single miRNA can regulate vascular integrity and angiogenesis, providing a new target for modulating vascular formation and function.

[1]  Ligang Wu,et al.  Let me count the ways: mechanisms of gene regulation by miRNAs and siRNAs. , 2008, Molecular cell.

[2]  C. Betsholtz,et al.  Pericyte‐specific expression of Rgs5: implications for PDGF and EDG receptor signaling during vascular maturation , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[3]  Holger Gerhardt,et al.  Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis , 2007, Nature.

[4]  J. Licht,et al.  Sprouty proteins: multifaceted negative-feedback regulators of receptor tyrosine kinase signaling. , 2006, Trends in cell biology.

[5]  Robo4 stabilizes the vascular network by inhibiting pathologic angiogenesis and endothelial hyperpermeability. , 2008, Nature medicine.

[6]  J. Klumperman,et al.  Egfl7 knockdown causes defects in the extension and junctional arrangements of endothelial cells during zebrafish vasculogenesis , 2008, Developmental dynamics : an official publication of the American Association of Anatomists.

[7]  Y. Maehara,et al.  Spreds Are Essential for Embryonic Lymphangiogenesis by Regulating Vascular Endothelial Growth Factor Receptor 3 Signaling , 2007, Molecular and Cellular Biology.

[8]  E. Dejana,et al.  Foxs and Ets in the transcriptional regulation of endothelial cell differentiation and angiogenesis. , 2007, Biochimica et biophysica acta.

[9]  J. Wood,et al.  Dissection of angiogenic signaling in zebrafish using a chemical genetic approach. , 2002, Cancer cell.

[10]  W. Cavenee,et al.  Platelet-derived growth factor-B enhances glioma angiogenesis by stimulating vascular endothelial growth factor expression in tumor endothelia and by promoting pericyte recruitment. , 2003, The American journal of pathology.

[11]  H. Lowman,et al.  Therapeutic anti-VEGF antibodies. , 2008, Handbook of experimental pharmacology.

[12]  Stefanie Dimmeler,et al.  Role of Dicer and Drosha for Endothelial MicroRNA Expression and Angiogenesis , 2007, Circulation research.

[13]  Yong Zhao,et al.  Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis , 2005, Nature.

[14]  R. Klein,et al.  Ephrin-B Reverse Signaling Is Mediated by a Novel PDZ-RGS Protein and Selectively Inhibits G Protein–Coupled Chemoattraction , 2001, Cell.

[15]  E. Mandelkow,et al.  Spred1 and TESK1--two new interaction partners of the kinase MARKK/TAO1 that link the microtubule and actin cytoskeleton. , 2008, Molecular biology of the cell.

[16]  Wigard P Kloosterman,et al.  In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes , 2005, Nature Methods.

[17]  A. Zeiher,et al.  Homeobox A9 Transcriptionally Regulates the EphB4 Receptor to Modulate Endothelial Cell Migration and Tube Formation , 2004, Circulation research.

[18]  D. Gorski,et al.  Regulation of angiogenesis through a microRNA (miR-130a) that down-regulates antiangiogenic homeobox genes GAX and HOXA5. , 2008, Blood.

[19]  R. Yeh,et al.  MicroRNA regulation of cell lineages in mouse and human embryonic stem cells. , 2008, Cell stem cell.

[20]  Michael Kertesz,et al.  The role of site accessibility in microRNA target recognition , 2007, Nature Genetics.

[21]  E. Butcher,et al.  Regulation of Chemotactic and Proadhesive Responses to Chemoattractant Receptors by RGS (Regulator of G-protein Signaling) Family Members* , 1998, The Journal of Biological Chemistry.

[22]  Burton B. Yang,et al.  MicroRNA-378 promotes cell survival, tumor growth, and angiogenesis by targeting SuFu and Fus-1 expression , 2007, Proceedings of the National Academy of Sciences.

[23]  R. Baron,et al.  Spred is a Sprouty-related suppressor of Ras signalling , 2001, Nature.

[24]  H. Horvitz,et al.  MicroRNA Expression in Zebrafish Embryonic Development , 2005, Science.

[25]  Michael T. McManus,et al.  Dysregulation of Cardiogenesis, Cardiac Conduction, and Cell Cycle in Mice Lacking miRNA-1-2 , 2007, Cell.

[26]  J. Klein,et al.  Positive and Negative Roles of p85α and p85β Regulatory Subunits of Phosphoinositide 3-Kinase in Insulin Signaling* , 2003, Journal of Biological Chemistry.

[27]  A. Yoshimura,et al.  The Sprouty-related protein, Spred, inhibits cell motility, metastasis, and Rho-mediated actin reorganization , 2004, Oncogene.

[28]  Norbert Perrimon,et al.  Functional screening identifies miR-315 as a potent activator of Wingless signaling , 2007, Proceedings of the National Academy of Sciences.

[29]  G. Yancopoulos,et al.  EphB ligand, ephrinB2, suppresses the VEGF‐ and angiopoietin‐1‐induced Ras/mitogen‐activated protein kinase pathway in venous endothelial cells , 2002, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[30]  J. Huot,et al.  Integrating the VEGF signals leading to actin-based motility in vascular endothelial cells. , 2000, Trends in cardiovascular medicine.

[31]  Laura Mariani,et al.  MicroRNAs modulate the angiogenic properties of HUVECs. , 2006, Blood.

[32]  Jordan S. Pober,et al.  Dicer Dependent MicroRNAs Regulate Gene Expression and Functions in Human Endothelial Cells , 2007, Circulation research.

[33]  Xuri Li,et al.  PDGF-D is a potent transforming and angiogenic growth factor , 2003, Oncogene.

[34]  C. Myers,et al.  Homeobox B3 Promotes Capillary Morphogenesis and Angiogenesis , 2000, The Journal of cell biology.

[35]  Kenneth P. Roos,et al.  Autocrine VEGF Signaling Is Required for Vascular Homeostasis , 2007, Cell.

[36]  Didier Y. R. Stainier,et al.  The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation , 2004, Nature.

[37]  H. Baier,et al.  A transgene-assisted genetic screen identifies essential regulators of vascular development in vertebrate embryos. , 2007, Developmental biology.

[38]  T. Curran,et al.  Cardiovascular and Craniofacial Defects in Crk-Null Mice , 2006, Molecular and Cellular Biology.

[39]  A. Yoshimura,et al.  Spred-1 Negatively Regulates Interleukin-3-mediated ERK/Mitogen-activated Protein (MAP) Kinase Activation in Hematopoietic Cells* , 2004, Journal of Biological Chemistry.

[40]  J. Fish,et al.  The Expression of Endothelial Nitric-oxide Synthase Is Controlled by a Cell-specific Histone Code* , 2005, Journal of Biological Chemistry.

[41]  George E. Sandusky,et al.  Dicer Is Required for Embryonic Angiogenesis during Mouse Development* , 2005, Journal of Biological Chemistry.

[42]  Ulf Eriksson,et al.  Angiogenesis stimulated by PDGF‐CC, a novel member in the PDGF family, involves activation of PDGFR‐aa and ‐ap receptors , 2002 .

[43]  W. Schiemann,et al.  Identification and characterization of regulator of G protein signaling 4 (RGS4) as a novel inhibitor of tubulogenesis: RGS4 inhibits mitogen-activated protein kinases and vascular endothelial growth factor signaling. , 2004, Molecular biology of the cell.

[44]  Joshua T. Mendell,et al.  MicroRNA-126 regulates endothelial expression of vascular cell adhesion molecule 1 , 2008, Proceedings of the National Academy of Sciences.

[45]  Yong Zhao,et al.  A developmental view of microRNA function. , 2007, Trends in biochemical sciences.