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

Abstract

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-α, 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-α 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-α induced miR-200b blunting proangiogenic functions of GATA2 and VEGFR2.

The MicroRNA miR-199a-5p Down-regulation Switches on Wound Angiogenesis by Derepressing the v-ets Erythroblastosis Virus E26 Oncogene Homolog 1-Matrix Metalloproteinase-1 Pathway

“…Cell and Cell Culture-Human dermal microvascular endothelial cells (HMECs) were grown in MCDB-131 medium supplemented with 10% FBS, 10 mM L-glutamine, 100 IU/ml penicillin, and 0.1 mg/ml streptomycin as described previously (2,3,5). Primary adult human dermal microvascular endothelial cells were cultured in EBM-2 medium (Lonza, Basel, Switzerland) supplemented with EGM-2MV single quotes as described by the manufacturer.…”

“…miR expression was determined by miR TaqMan assays and TaqMan microRNA reverse transcription kit, followed by quantitative real time PCR using Universal PCR Master Mix (Applied Biosystems) (3,5). For mRNA expression studies, total cDNA synthesis was achieved by a SuperScript TM III first strand synthesis system (Applied Biosystems).…”

“…For mRNA expression studies, total cDNA synthesis was achieved by a SuperScript TM III first strand synthesis system (Applied Biosystems). The transcription levels of Ets-1, MMP-1, MMP-3, and MMP-9 were assessed by real time PCR using SYBR green-I (Invitrogen) as described previously (3,5). The sequences of primers are shown in supplemental Table S1.…”

Dicer Knockdown Inhibits Endothelial Cell Tumor Growth via MicroRNA 21a-3p Targeting of Nox-4

“…RNA Extraction and Quantitative Real Time PCR-For detection of mRNA and miRNA, total RNA from tissue and cells was extracted using miRVana miRNA isolation kit according to the manufacturer's protocol (Ambion/Invitrogen) (15,16). For pre-miRNA isolation, RNA were collected with two column filtration procedure using miRVana miRNA isolation kit according to the manufacturer's protocol (18).…”

“…For mRNA expression studies, cDNA synthesis was achieved by SuperScript TM III first strand synthesis system (Applied Biosystems/Invitrogen). The abundance of mRNA for genes of interest was quantified by using real time PCR with double-stranded DNA binding dye SYBR green-I (15,16). The primer sets for JE/MCP-1, nox-1-4, duox1, and duox2 were the same sets as previously reported (8).…”