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.

Conclusions: 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.

Figure 1

Acute transient downregulation of endothelial microRNA-200b (miR-200b) was essential in supporting cutaneous wound angiogenesis. Quantitative polymerase chain reaction (PCR) analysis of miR-200b expression of (A) days 3, 7, and 14 wound-edge tissue (8 × 16 mm full-thickness excisional wounds, n = 6) and (B) laser microdissected endothelial tissue element (n = 4) from day 3 wounds, compared with their respective control skin sample. Control or miR-200b overexpressing lentiviral particle (2 × 10⁷ cfu/mL, 50 μL per site) was intradermally delivered to the skin. Three days after delivery of lentivirus, excisional wounds were created using 6-mm diameter punches as described in the Methods section. C, Wound closure was monitored on days 1, 3, 5, and 7 postwounding after treatment with miR-200b or control miR overexpressing lentiviral particle by digital planimetry and was presented as percentage of wound closure (n = 8). Wound angiogenesis, as depicted by (D) laser Doppler (n = 6) and (E) quantification of CD31 positive staining (n = 7), of the cutaneous wound was analyzed on day 7 postwounding. Results are mean ± SEM. ***P < 0.001, **P < 0.01, *P < 0.05 compared with respective control. LCM indicates laser capture microdissection.

Figure 2

MicroRNA-200b (miR-200b) modulated antiangiogenic effects by targeting globin transcription factor binding protein 2 (GATA2) and vascular endothelial growth factor receptor 2 (VEGFR2). miR target reporter luciferase assay using construct with either (A) GATA2 3′ untranslated region (3′UTR) or (B) VEGFR2 3′UTR in miR-200b mimic delivered human embryonic kidney 293 cells (HEK-293 cells). Results were normalized with renilla luciferase activity (n = 4). *P < 0.05 compared with control mimic transfected cells, ⁺⁺P < 0.01 compared with respective wild-type plasmid-transfected cells. Western blot analysis of (C) GATA2 and (D) VEGFR2 protein expression in miR-200b mimic delivered human dermal microvascular endothelial cells (HDMECs; n = 4). β-actin serves as loading control. Matrigel tube formation at 8 hours in HDMEC treated with control mimic, miR-200b mimic, and miR-200b with either (E) GATA2 or (F) VEGFR2 overexpressing lentivirus (n = 4). ***P < 0.001, **P < 0.01, *P < 0.05 compared with respective control; ⁺P < 0.05, ⁺⁺P < 0.01 compared with miR-200b mimic alone. GFP indicates green fluorescent protein.

Figure 3

Expression of endothelial globin transcription factor binding protein 2 (GATA2) and vascular endothelial growth factor receptor 2 (VEGFR2) was induced during wound healing process and was modulated by microRNA-200b (miR-200b). Representative diagram shows (A) GATA2 and (B) VEGFR2 immunohistochemistry (red), in the intact skin, wound sample from days 3 and 7 postwounding. Representative diagram shows (C) GATA2 and (D) VEGFR2 immunohistochemistry (red) in day 7 wound sample treated with control or miR-200b overexpressing lentivirus. Colocalization of the GATA2 or VEGFR2 signaling with endothelial marker CD31 (green) was achieved by coincubation of anti-CD31 and anti-GATA2 or anti-VEGFR2 antibodies. Quantification of GATA2 or VEGFR2 intensity in CD31-positive cells. Results are mean ± SEM (n = 4). ***P < 0.001, **P < 0.01 compared with respective control; ⁺P < 0.05 compared with day 3 wounds.

Figure 4

Diabetic wounds exhibited disrupted microRNA-200b (miR-200b)-globin transcription factor binding protein 2 (GATA2)-vascular endothelial growth factor receptor 2 (VEGFR2) signaling. A, Quantitative polymerase chain reaction (PCR) analysis of miR-200b expression of day 3 wound-edge tissues isolated from m+/db nondiabetic mice and db/db diabetic mice. Fold changes of the wound samples were compared with their corresponding intact skin control. Results are mean ± SEM (n = 5). ***P < 0.001, ⁺P < 0.05 compared with m+/db d3 wounds. Representative diagram shows (B) GATA2 and (C) VEGFR2 immunohistochemistry (red) in the day 7 wound sample from m+/db nondiabetic mice and db/db diabetic mice. Colocalization of the GATA2 or VEGFR2 signaling with endothelial marker CD31 (green) was achieved by coincubation of anti-CD31 and anti-GATA2 or anti-VEGFR2 antibodies. Quantification of GATA2 or VEGFR2 intensity in CD31-positive cells. Results are mean ± SEM (n = 4). **P < 0.01, *P < 0.05 compared with day 7 m+/db nondiabetic control mice.

Figure 5

MicroRNA-200b (miR-200b) served as a mediator of tumor necrosis factor-α (TNF-α)–induced downregulation of globin transcription factor binding protein 2 (GATA2) and vascular endothelial growth factor receptor 2 (VEGFR2) expression, and antiangiogenic response of endothelial cells. A, Quantitative polymerase chain reaction (PCR) analysis of miR-200b expression after treatment of TNF-α as indicated time in human dermal microvascular endothelial cells (HDMECs). B, Western blot analysis of GATA2 and VEGFR2 protein expression in TNF-α-treated HDMECs at different time points. C, Western blot analysis of GATA2 and VEGFR2 protein expression in TNF-α-treated HDMECs in the presence or absence of anti-miR-200b. β-actin serves as loading control. D, Matrigel tube formation visualized by phase contrast microscopy at 8 hours in TNF-α-treated HDMECs in the presence or absence of anti-miR-200b. Results are mean ± SEM (n = 4). ***P < 0.001, **P < 0.01, *P < 0.05 compared with respective control; ⁺⁺P < 0.01, ⁺P < 0.05 compared with cells treated with TNF alone. NS indicates not statistically significant.

Figure 6

Neutralization of tumor necrosis factor-α (TNF-α) reversed the aberrant microRNA-200b (miR-200b) induction, rescued the loss of globin transcription factor binding protein 2 (GATA2) and vascular endothelial growth factor receptor 2 (VEGFR2) expression, and improved wound closure and angiogenesis. A, Quantitative polymerase chain reaction (PCR) analysis of miR-200b expression of d3 wound-edge tissue isolated from m+/db nondiabetic mice, db/db diabetic mice treated with placebo, and db/db diabetic mice treated with soluble TNF receptor 1 (sTNFR1; n = 5). Fold changes of the wound samples were compared with their corresponding intact skin control. B, Quantification of GATA2 or VEGFR2 intensity in CD31 positive cells (n = 4). C, Representative diagram shows GATA2 and VEGFR2 immunohistochemistry (red) in the day 7 wound sample from m+/db nondiabetic mice, db/db diabetic mice treated with placebo, and db/db diabetic mice treated with sTNFR1. Colocalization of the GATA2 or VEGFR2 signaling with endothelial marker CD31 (green) was achieved by coincubation of anti-CD31 and anti-GATA2 or anti-VEGFR2 antibodies. D, Wound closure was monitored on days 1, 3, 5, and 7 postwounding and was presented as percentage of wound closure (n = 6): black, m+/db nondiabetic mice; red, db/db diabetic mice treated with placebo; and blue, db/db diabetic mice treated with sTNFR1. Wound angiogenesis, as depicted by (E) cutaneous blood flow measured by laser Doppler (n = 6) and (F) quantification of CD31 positive staining (n = 4) on day 7 postwounding. Results are mean ± SEM. ***P < 0.001, *P < 0.05 compared with m+/db nondiabetic wounds; ⁺⁺P < 0.01, ⁺P < 0.05 compared with db/db diabetic wounds.