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

Abstract

miR-199a-5p plays a critical role in controlling cardiomyocyte survival. However, its significance in endothelial cell biology remains ambiguous. Here, we report the first evidence that miR-199a-5p negatively regulates angiogenic responses by directly targeting v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets-1). Induction of miR-199a-5p in human dermal microvascular endothelial cells (HMECs) blocked angiogenic response in Matrigel® culture, whereas miR-199a-5p-deprived cells exhibited enhanced angiogenesis in vitro. Bioinformatics prediction and miR target reporter assay recognized Ets-1 as a novel direct target of miR-199a-5p. Delivery of miR-199a-5p blocked Ets-1 expression in HMECs, whereas knockdown endogenous miR-199a-5p induced Ets-1 expression. Matrix metalloproteinase 1 (MMP-1), one of the Ets-1 downstream mediators, was negatively regulated by miR-199a-5p. Overexpression of Ets-1 not only rescued miR-199a-5p-dependent anti-angiogenic effects but also reversed miR-199a-5p-induced loss of MMP-1 expression. Similarly, Ets-1 knockdown blunted angiogenic response and induction of MMP-1 in miR-199a-5p-deprived HMECs. Examination of cutaneous wound dermal tissue revealed a significant down-regulation of miR-199a-5p expression, which was associated with induction of Ets-1 and MMP-1. Mice carrying homozygous deletions in the Ets-1 gene exhibited blunted wound blood flow and reduced abundance of endothelial cells. Impaired wound angiogenesis was associated with compromised wound closure, insufficient granulation tissue formation, and blunted induction of MMP-1. Thus, down-regulation of miR-199a-5p is involved in the induction of wound angiogenesis through derepressing of the Ets-1-MMP1 pathway.

FIGURE 1.

miR-199a-5p induces angiostatic effects in HMECs. A and B, quantitative real time PCR analysis of miR-199a-5p expression after delivery of miR-199a-5p mimic (A) or miR-199a-5p inhibitor (B). C and D, HMECs were subjected to Matrigel® culture after transfection of miR-199a-5p mimic (C) or miR-199a-5p inhibitor (D). Images of tube formation were visualized and captured by phase contrast microscopy. Representative images are shown from at least three independent experiments. Scale bar, 200 μm. Quantification of length of tube formation (percentage of control) of miR-199a-5p mimic or miR-199a-5p inhibitor-delivered cells is shown. The results are the means ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with control.

FIGURE 2.

Ets-1 serves as a bona fide miR-199a-5p target. A, in silico study revealing a possible binding site in Ets-1 3′-UTR (at positions 2708–2714) for miR-199a-5p as predicted by Targetscan, Pictar, MiRanda, and miRDB. B, miR target reporter luciferase assay after miR-199a-5p mimic delivery in HEK-293 cells using wild type pLu-wild type Ets-1–3UTR plasmid or pLu-mutated Ets-1–3UTR plasmid. Open bars and solid bars represent control mimic and miR-199a-5p mimic delivered cells, respectively. The results were normalized with data obtained from assay with Renilla luciferase. C and D, Western blot analysis of Ets-1 protein expression in miR-199a-5p mimic delivered (C) or depleted (D) HMECs. β-Actin serves a loading control. Representative blots from three independent experiments and quantification of band intensity relative to control are presented. E, representative images showing Ets-1 protein expression (green) after miR-199a-5p mimic delivery from three independent experiments. Nuclear counterstain with DAPI (blue), actin staining with phalloidin (red), and the corresponding merged image are shown in the bottom panels. The results are the means ± S.E. ***, p < 0.001; *, p < 0.05 compared with corresponding control; +++, p < 0.001 compared with pLu-wild type Ets-1–3′-UTR plasmid transfected cells.

FIGURE 3.

Expression of MMP-1 in Ets-1 knock-down, miR-199a-5p mimic delivered and miR-199a-5p depleted HMECs. Real time PCR analysis of MMP-1 expression after transfection of Ets-1 siRNA (A), miR-199a-5p mimic (B), or miR-199a-5p inhibitor (C) is shown. The results are the means ± S.E. *, p < 0.05 compared with corresponding control.

FIGURE 4.

Ets-1 overexpression or knockdown reversed miR-199a-5p-induced angiogenic control and its associated regulation of MMP-1 gene expression. A and B, Matrigel® tube formation visualized by phase contrast microscopy at 8 h after delivery of control or miR-199a-5p mimic in the presence or absence of Ets-1 pcDNA (A) or control or miR-199a-5p inhibitor in the presence or absence of Ets-1 siRNA (B). Representative images are shown from three independent experiments. Scale bar, 200 μm. The bar graphs indicate quantification of length of tube formation (percentage of control). C and D, real time PCR analysis of MMP-1 expression after delivery of control or miR-199a-5p mimic in the presence or absence of Ets-1 pcDNA (C) or control or miR-199a-5p inhibitor in the presence or absence of Ets-1 siRNA (D). The results are the means ± S.E. **, p < 0.01 compared with control mimic/inhibitor; +++, p < 0.001; ++, p < 0.01 compared with miR-199a-5p mimic + pcDNA/miR-199a-5p + control siRNA.

FIGURE 5.

Down-regulation of dermal and endothelial miR-199a-5p expression and induction of Ets-1 and MMP-1 in murine cutaneous wound edge tissue. A and B, real time PCR analysis of miR-199a-5p expression in laser-captured microdissected dermis (A, n = 4) or endothelial cells (B, n = 3) from intact skin or day 3 wound edge tissues (Epi, epidermis; Der, dermis). C, representative diagram shows Ets-1 immunohistochemistry (red) in the intact skin and wound sample from days 3 and 7 postwounding (n = 4). Co-localization of the Ets-1 signal with endothelial marker CD31 (green) was achieved by co-incubation of anti-CD31 and anti-Ets-1 antibodies, counterstained with DAPI (blue). D, bar graph indicates quantification of Ets-1 intensity in CD31 positive cells. E, Western blot analysis of MMP-1 protein expression in skin and day 3 and 7 wound tissue from C57BL/6 mice. β-Actin serves as loading control. Representative blots from three independent experiments and quantification of band intensity relative to control are presented (n = 3). The results are the means ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with skin.

FIGURE 6.

Ets-1 knock-out mice exhibit impaired wound closure. A, representative gel photo of genotyping PCR showing the presence of Ets-1 null band (250 bp) in PCR product from Ets-1^−/− tissue. PCR product from Ets-1^+/− mouse tissue reveals the presence of both wild type (200 bp) and null band, whereas Ets-1^+/+ mice contain only the wild type band. B, Western blot analysis of Ets-1 protein expression in skin tissue from Ets-1^+/+, Ets-1^+/−, and Ets-1^−/− mice. β-Actin serves as a loading control. Representative blots from three independent experiments and quantification of band intensity relative to control are presented (n = 4). C, wound closure was monitored on day (d) 1, 3, 5, 7, and 9 postwounding in Ets-1^+/+, Ets-1^+/−, and Ets-1^−/− mice by digital planimetry and was presented as a percentage of wound closure (n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with Ets-1^+/+ mice; +, p < 0.05; ++, p < 0.01 compared with Ets-1^+/− mice.

FIGURE 7.

Histological analyses of cutaneous wound edge tissue from Ets-1 knock-out mice. Representative images of hematoxylin-eosin (A) and Masson trichrome staining (B) of d9 wound tissue from Ets-1^+/+, Ets-1^+/−, and Ets-1^−/− mice are shown. Both hematoxylin-eosin and Masson's trichrome staining were performed on wound tissue sections obtained from three different wounds from wild type, heterozygous, and homozygous knock-out animals.

FIGURE 8.

Ets-1 knock-out mice exhibit impaired wound angiogenesis. A, representative laser Doppler image of day 7 wound tissue from Ets-1^+/+, Ets-1^+/−, and Ets-1^−/− mice. Quantification of blood flow in the wound edge area is presented as the mean ± S.E. (n = 5). B, representative images from immunostaining of CD31 from day 7 wound tissue from Ets-1^+/+, Ets-1^+/−, and Ets-1^−/− mice. The bar graph represents quantification of CD31 positive area in the wound area (n = 4). C, Western blot analysis of MMP-1 protein expression in day 7 wound tissue from Ets-1^+/+, Ets-1^+/−, and Ets-1^−/− mice. β-Actin serves as loading control. Representative blots from three independent experiments and quantification of band intensity relative to control are presented (n = 3). **, p < 0.01; *, p < 0.05 compared with Ets-1^+/+ mice; +, p < 0.05 compared with Ets-1^+/− mice.