Downregulation of Endothelial MicroRNA-200b Supports Cutaneous Wound Angiogenesis By Desilencing GATA Binding Protein 2 and Vascular Endothelial Growth Factor Receptor 2

Objective—MicroRNAs (miRs) regulate angiogenesis by posttranscriptional silencing of target genes. The significance of angiostatic miR-200b in switching on skin wound angiogenesis was tested. Methods and Results—Wounding caused imminent and transient downregulation of miR-200b in dermal wound-edge endothelial cells. Derailing this injury response by lentiviral delivery of miR-200b in vivo impaired wound angiogenesis. Computational prediction, target reporter luciferase assay, and Western blot analysis provided first evidence that miR-200b targets globin transcription factor binding protein 2 (GATA2) and vascular endothelial growth factor receptor 2 (VEGFR2). Overexpression of GATA2 or VEGFR2 in endothelial cells rescued the angiostatic effect of miR-200b in vitro. Downregulation of miR-200b derepressed GATA2 and VEGFR2 expression to switch on wound angiogenesis, which was disrupted in diabetic wounds. Treatment of endothelial cells with tumor necrosis factor-&agr;, a proinflammatory cytokine abundant in diabetic wounds, induced miR-200b expression, silenced GATA2 and VEGFR2, and suppressed angiogenesis. These outcomes were attenuated using anti-miR-200b strategy. Neutralization of tumor necrosis factor-&agr; in the diabetic wounds improved wound angiogenesis and closure, which was accompanied by downregulation of miR-200b expression and desilencing of GATA2 and VEGFR2. Conclusion—Injury-induced repression of miR-200b turned on wound angiogenesis. In mice with diabetes mellitus,excessive tumor necrosis factor-&agr; induced miR-200b blunting proangiogenic functions of GATA2 and VEGFR2.

[1]  G. Goodall,et al.  The Notch ligand Jagged2 promotes lung adenocarcinoma metastasis through a miR-200-dependent pathway in mice. , 2011, The Journal of clinical investigation.

[2]  R. Stephens,et al.  Downregulated MicroRNA-200a in Meningiomas Promotes Tumor Growth by Reducing E-Cadherin and Activating the Wnt/β-Catenin Signaling Pathway , 2009, Molecular and Cellular Biology.

[3]  G. Gordillo,et al.  Micromanaging Vascular Biology: Tiny MicroRNAs Play Big Band , 2009, Journal of Vascular Research.

[4]  P. Waterhouse,et al.  miR-451 regulates zebrafish erythroid maturation in vivo via its target gata2. , 2009, Blood.

[5]  Gerry Rayman,et al.  Neurovascular Factors in Wound Healing in the Foot Skin of Type 2 Diabetic Subjects , 2008, Diabetes Care.

[6]  B. Black,et al.  Transcriptional control of endothelial cell development. , 2009, Developmental cell.

[7]  R. Qian,et al.  MicroRNA‐320 EXPRESSION IN MYOCARDIAL MICROVASCULAR ENDOTHELIAL CELLS AND ITS RELATIONSHIP WITH INSULIN‐LIKE GROWTH FACTOR‐1 IN TYPE 2 DIABETIC RATS , 2009, Clinical and experimental pharmacology & physiology.

[8]  L. Debusk,et al.  Dual functional roles of Tie-2/angiopoietin in TNF-α-mediated angiogenesis , 2004 .

[9]  C. Sen,et al.  Evidence for the Involvement of miRNA in Redox Regulated Angiogenic Response of Human Microvascular Endothelial Cells , 2008, Arteriosclerosis, thrombosis, and vascular biology.

[10]  D. Greenhalgh,et al.  Expression and localization of p53 and bcl‐2 in healing wounds in diabetic and nondiabetic mice , 2008, Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society.

[11]  X. Han,et al.  Transcriptional Up-regulation of Endothelial Cell Matrix Metalloproteinase-2 in Response to Extracellular Cues Involves GATA-2* , 2003, Journal of Biological Chemistry.

[12]  Jiri Zavadil,et al.  Epigenetic silencing of the oncogenic miR-17-92 cluster during PU.1-directed macrophage differentiation , 2011, The EMBO journal.

[13]  J. Miyazaki,et al.  Interaction between Hex and GATA Transcription Factors in Vascular Endothelial Cells Inhibits flk-1/KDR-mediated Vascular Endothelial Growth Factor Signaling* , 2004, Journal of Biological Chemistry.

[14]  Christopher C W Hughes,et al.  TNF primes endothelial cells for angiogenic sprouting by inducing a tip cell phenotype. , 2008, Blood.

[15]  J. Long,et al.  Identification of MicroRNA-93 as a Novel Regulator of Vascular Endothelial Growth Factor in Hyperglycemic Conditions* , 2010, The Journal of Biological Chemistry.

[16]  H. Aburatani,et al.  Epigenetically coordinated GATA2 binding is necessary for endothelium‐specific endomucin expression , 2011, The EMBO journal.

[17]  M. Herlyn,et al.  Adenoviral mediated gene transfer of PDGF‐B enhances wound healing in type I and type II diabetic wounds , 2004, Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society.

[18]  Tae Jin Lee,et al.  p53 regulates epithelial–mesenchymal transition through microRNAs targeting ZEB1 and ZEB2 , 2011, The Journal of experimental medicine.

[19]  D. Graves,et al.  Impaired wound healing in mouse models of diabetes is mediated by TNF-α dysregulation and associated with enhanced activation of forkhead box O1 (FOXO1) , 2010, Diabetologia.

[20]  L. Debusk,et al.  Dual functional roles of Tie-2/angiopoietin in TNF-alpha-mediated angiogenesis. , 2004, American journal of physiology. Heart and circulatory physiology.

[21]  Marcus Fruttiger,et al.  The Notch Ligands Dll4 and Jagged1 Have Opposing Effects on Angiogenesis , 2009, Cell.

[22]  E. Collard,et al.  Macrophage Dysfunction Impairs Resolution of Inflammation in the Wounds of Diabetic Mice , 2010, PloS one.

[23]  Chawnshang Chang,et al.  Monocyte/macrophage androgen receptor suppresses cutaneous wound healing in mice by enhancing local TNF-alpha expression. , 2009, The Journal of clinical investigation.

[24]  C. Sen,et al.  miR-200b Targets Ets-1 and Is Down-regulated by Hypoxia to Induce Angiogenic Response of Endothelial Cells* , 2010, The Journal of Biological Chemistry.

[25]  G. Condorelli,et al.  Deregulation of microRNA-503 Contributes to Diabetes Mellitus–Induced Impairment of Endothelial Function and Reparative Angiogenesis After Limb Ischemia , 2011, Circulation.

[26]  Donald E. Ingber,et al.  A mechanosensitive transcriptional mechanism that controls angiogenesis , 2009, Nature.

[27]  C. Sen,et al.  Characterization of the acute temporal changes in excisional murine cutaneous wound inflammation by screening of the wound-edge transcriptome. , 2008, Physiological genomics.

[28]  C. Attinger,et al.  Functional Reconstruction of the Diabetic Foot , 2010, Seminars in plastic surgery.

[29]  Avner Friedman,et al.  Hypoxia inducible microRNA 210 attenuates keratinocyte proliferation and impairs closure in a murine model of ischemic wounds , 2010, Proceedings of the National Academy of Sciences.

[30]  E. Dejana,et al.  Endothelial cadherins and tumor angiogenesis. , 2006, Experimental cell research.

[31]  Simone Brabletz,et al.  The ZEB1/miR‐200 feedback loop controls Notch signalling in cancer cells , 2011, The EMBO journal.

[32]  Jaeseung Yoon,et al.  Regulation of vascular endothelial growth factor signaling by miR-200b , 2011, Molecules and cells.

[33]  T. K. Hunt,et al.  Human skin wounds: A major and snowballing threat to public health and the economy , 2009, Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society.

[34]  D. Keene,et al.  Intradermal injection of lentiviral vectors corrects regenerated human dystrophic epidermolysis bullosa skin tissue in vivo. , 2004, Molecular therapy : the journal of the American Society of Gene Therapy.

[35]  A. Friedman,et al.  Transcriptome-wide analysis of blood vessels laser captured from human skin and chronic wound-edge tissue , 2007, Proceedings of the National Academy of Sciences.

[36]  Subrata Chakrabarti,et al.  MicroRNA-200b Regulates Vascular Endothelial Growth Factor–Mediated Alterations in Diabetic Retinopathy , 2011, Diabetes.

[37]  Pierre Roux,et al.  TNFα induces sequential activation of Cdc42- and p38/p53-dependent pathways that antagonistically regulate filopodia formation , 2004, Journal of Cell Science.

[38]  K. Tsuchida,et al.  Intramuscular Gene Transfer of Soluble Tumor Necrosis Factor-&agr; Receptor 1 Activates Vascular Endothelial Growth Factor Receptor and Accelerates Angiogenesis in a Rat Model of Hindlimb Ischemia , 2004, Circulation.

[39]  D. Accili,et al.  Glucose effects on skin keratinocytes: implications for diabetes skin complications. , 2001, Diabetes.

[40]  Simone Brabletz,et al.  The ZEB/miR‐200 feedback loop—a motor of cellular plasticity in development and cancer? , 2010, EMBO reports.

[41]  G. Ligresti,et al.  Macrophage-Derived Tumor Necrosis Factor-&agr; Is an Early Component of the Molecular Cascade Leading to Angiogenesis in Response to Aortic Injury , 2011, Arteriosclerosis, thrombosis, and vascular biology.

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

[43]  T. Tuschl,et al.  MicroRNA-24 Regulates Vascularity After Myocardial Infarction , 2011, Circulation.