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*

Background: The role of miR-199a-5p in angiogenesis remains unclear. Results: miR-199a-5p exerts angiostatic effects by targeting Ets-1-MMP1 pathway and is down-regulated in skin wound healing. Conclusion: Down-regulation of miR-199a-5p switches on wound angiogenesis through derepressing of Ets-1-MMP1 pathway. Significance: This investigation provides novel mechanistic insight explaining miR-dependent regulation of wound angiogenesis and the foundation of developing therapeutic intervention in treating chronic nonhealing wounds. 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.

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-5pdeprived 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.
Angiogenesis, sprouting of pre-existing blood vessels, is one of the major biological responses supporting both cutaneous wound healing and tumorogenesis. Emerging evidence has revealed that microRNAs (miRs) 2 play a critical role in regulation of angiogenesis (1,2). These small RNA exert substantial gene regulatory effects by a number of ways including physical interaction with the 3Ј-UTR of mRNA, hindering translation or leading to transcript degradation. Our group and others have characterized the significance of specific miRs in modulating the angiogenic response. miR-210, for example, supports angiogenesis by targeting angiostatic proteins ephrin-A3 and protein-tyrosine phosphatase 1B (1). miR-200b, a hypoxia-repressible miR (3), negatively regulates angiogenic response by silencing vascular endothelial growth factor (4), GATA-binding protein 2 (5), and vascular endothelial growth factor receptor 2 (5).

EXPERIMENTAL PROCEDURES
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. HEK-293 cells were maintained with high glucose DMEM supplemented with 10% FBS and 100 IU/ml penicillin, 0.1 mg/ml streptomycin. All of the cells were cultured in standard culture conditions (at 37°C with 20% O 2 and 5% CO 2 ). The cells were seeded in a 12-well plate at a density of 0.12 ϫ 10 6 cells/well 24 h before treatment. Delivery of small RNAs was achieved using DharmaFECTTM 1 transfection reagent (Dharmacon RNA Technologies, Denver, CO). miR-199a-5p mimic (50 nM), miR-199a-5p inhibitor (100 nM), or siRNA smart pool for human Ets-1 or MMP-1 (100 nM) were obtained from Dharmacon RNA Technologies. Unless specific, the cells were lysed after 72 h (HMECs and HEK-293) or 48 h (primary endothelial cells) of transfection and RNA/protein were collected for gene expression study. For Ets-1 overexpression studies, expressing plasmid encoding Ets-1 (Ets-1 pcDNA), a generous gift from Dr. Michael C. Ostrowski (The Ohio State University, Columbus, OH) (17) (1 g/well), was delivered to the endothelial cells using Lipofectamine TM LTX/plus reagent as described previously (3). Two days after the plasmid delivery, the cells were subjected to miR-199a-5p mimic (or control mimic) delivery as described above for another 48 h for Matrigel analysis, and RNA/protein extraction. For miR-199a-5p inhibitor-Ets-1 siRNA co-transfection study, both miR-199a-5p inhibitor and Ets-1 siRNA (or control siRNA) were mixed together and incubated with Dharma-FECTTM 1 transfection reagent. For 72 h, the gene-liposome complex was delivered to endothelial cells that were subjected to in vitro angiogenesis studies or for RNA/protein extraction.
Mice and Secondary Intention Excisional Murine Dermal Wound Model-Male C57BL/6 mice (age, 8 -10 weeks old) were obtained from Harlan Laboratory. The Ets-1-deficient mice (129/Sv ϫ C57BL/6 ϫ FVB/N) were kindly provided by Dr. Michael C. Ostrowski. Genotyping was performed with DNA from tail biopsies by PCR using specific primers (supplemental Table S1) with the following protocol: 94°C for 45 s, 58°C for 45 s, and 72°C for 1 min (40 cycles). Two or four 6-mm diameter full thickness excisional wounds were developed on the dorsal skin of mice with a 6-mm disposable biopsy punch. A surgical splinted wound model was employed for the wound closure study, histological analysis, and collagen deposition assessment as described previously (18). Briefly, a donutshaped splint with a 8-mm inner diameter was created from a 0.5-mm-thick silicone sheet (Grace Bio-Laboratories, Bend, OR) and placed such that the wound was centered within the splint. To fix the splint to the skin, an immediate bonding adhe-sive was used, followed by interrupted 5-0 nylon sutures (Ethicon, Inc., Somerville, NJ) to maintain position. All of the animal studies were performed in accordance with protocols approved by the Laboratory Animal Care and Use Committee of the Ohio State University. During the wounding procedure, the mice were anesthetized by low dose isoflurane inhalation for 5-10 min/standard recommendation. Each wound was digitally photographed at the time point indicated. Wound size was calculated by the software Image J. The animals were sacrificed at the indicated postwounding time point, and wound tissues were harvested (as frozen, in optimal cutting temperature compound or fixed in buffered formalin) for further analysis.
RNA Isolation and Quantitative Real Time PCR-The miR-Vana miRNA isolation kit was employed to extract total RNA from tissue and cells according to the manufacturer's protocol (Ambion). 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). 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.
miR Target Reporter Luciferase Assay-pLu-wild type Ets1-3Ј-UTR plasmid or construct carrying the mutation of the predicted miR-199a-5p binding site in Ets1-3Ј-UTR (100 ng) (Signosis, Sunnyvale, CA) was delivered to HEK-293 cells using Lipofectamine TM LTX/plus reagent (Invitrogen) according to the manufacturer's protocol. The constructs were designed based on the sequence of miR-199a-5p binding sites and a total of 300 bp (starting from positions 2551-2850 of Ets-1 3Ј-UTR) was cloned in the 3Ј-UTR of constitutively active firefly luciferase construct. Mutation was made in the predicted miR-199a-5p binding site (from ACACUGG to UGUGACC, position 2708 -2714 of Ets-1 3Ј-UTR). Constitutively active Renilla luciferase plasmid (10 ng) was delivered to cells for normalization. The cell lysates were assayed with dual luciferase reporter assay kit (Promega, Madison, WI). The data are presented as ratio of firefly to Renilla luciferase activity as described previously (3,5).
Matrigel Assay-In vitro angiogenesis assay were assessed by tube formation ability on Matrigel culture as described previously (3,5). Endothelial cells were seeded on Matrigel precoated plate at a density of 5 ϫ 10 4 cells/well. The angiogenic ability was assessed 8 h (HMECs) or 24 h (primary endothelial cells) after plating on Matrigel, and the tube length was measured using the software AxioVision Rel 4.6 (Zeiss) (3,5).
Cell Migration Assay-Cell migration was determined using culture insert (Ibidi, Verona, WI) according to the manufacturer's instruction. Briefly, the cells were seeded at the confluent monolayer on the chambers of the culture insert. After seeding for 1 h, the insert was removed, and the cells were allowed to migrate. Cell migratory distance was measured 4 h after removal of insert using AxioVision Rel 4.6 software (Zeiss).
Cell Proliferation-Transfected cells were seeded in 96-well plate, and cell proliferation was determined by the CyQUANT cell proliferation assays (Invitrogen) as described previously (19).
Histological Analyses-Hematoxylin-eosin and Masson's trichrome staining were performed as described by our group previously (21). Briefly, tissue was collected and fixed in 10% buffered formalin solution for 2 weeks, after which they were processed for dehydration and paraffin embedding. Paraffinembedded tissue was sectioned at 5 m and processed for hematoxylin-eosin and Masson's trichrome staining.
Laser Capture Microdissection (LCM)-LCM was performed using the laser microdissection system from PALM Technologies (Bernreid, Germany) containing a PALM MicroBeam and RoboStage for high throughput sample collection and a PALM RoboMover (PALM Robo software, version 2.2) as described previously (5). For immunofluorescence-directed LCM, blood vessels were stained with CD31 antibody (1:25), FITC-conjugated secondary antibody (1:200), and subsequently visualized using fluorescent lamp. For dermal LCM, dermal fraction was identified based on the histology after hematoxylin staining. Endothelial or dermal tissue elements were isolated and captured under a 20ϫ ocular lens, using cut elements. Approximately 150,000 m 2 of tissue area was captured into lysis extraction buffer, and the extract was then held at Ϫ80°C for extraction process (5).
Laser Doppler-Dermal blood flow was analyzed by laser Doppler imager as described previously (5). The MoorLDI-Mark 2 laser Doppler blood perfusion imager (resolution, 256 ϫ 256 pixels; visible red laser beam at 633 nm) was used for mapping tissue blood flow under isofuran anesthetization.
Statistical Analyses-The data reported represent the means Ϯ S.E. Difference between two means was tested by Student t test, whereas one-way analysis of variance analysis was employed to compare three groups or more. p Ͻ 0.05 was considered statistically significant.

RESULTS
miR-199a-5p Is Angiostatic in HMECs-To determine the role of miR-199a-5p specifically on the angiogenic response of endothelial cells in vitro, miR-199a-5p mimic was transiently delivered to HMECs, and Matrigel tube formation was analyzed. Delivery of miR-199a-5p to the endothelial cells not only led to significant accumulation of miR-199a-5p ( Fig. 1A) but also exerted potent angiostatic effects (80% down-regulation compared with control) (Fig. 1C). Treatment of miR-199a-5p hairpin inhibitor blocked the endogenous expression of miR-199a-5p (Fig. 1B). Down-regulation of miR-199a-5p was accompanied by significant induction of tube formation ability by 40% in Matrigel culture (Fig. 1D). To investigate whether miR-199a-5p exerts general angiostatic effect on endothelial cells, we examine the effect of miR-199a-5p mimic or inhibitor on primary adult human dermal microvascular endothelial cells. Delivery of miR-199a-5p mimic to primary endothelial cells significantly attenuated the tube formation ability on Matrigel (supplemental Fig. S1, A and B), which was comparable with that on HMECs. Similarly, depletion of miR-199a-5p in primary endothelial cells significantly resulted in enhancement of angiogenic response. (supplemental Fig. S1, C and D). Consistent with the Matrigel study, delivery of miR-199a-5p mitigates endothelial cell migration (supplemental Fig. S2A). miR-199a-5p does not exert any effect on endothelial cell proliferation (supplemental Fig. S2B).
Murine Cutaneous Wound Edge Tissue Exhibited Decreased miR-199a-5p Expression, Ets-1 Induction, and Up-regulation of MMP-1-To connect the aforementioned observations to in vivo angiogenesis, we asked whether the miR-199a-5p-Ets-1-MMP-1 pathway takes place during postnatal angiogenesis such as during cutaneous wound healing. Our group previously reported that the miR-dependent angiogenic signals emerge miR-199a-5p Targets Ets-1 NOVEMBER 30, 2012 • VOLUME 287 • NUMBER 49 during inflammatory phase (5). We thus studied the expression level of miR-199a-5p day 3 postwounding in the excisional cutanous wound model. Data from dermal miR expression studies revealed a down-regulation of miR-199a-5p in response to wounding compared with intact skin in day 3 (Fig. 5A). These data are further supported by the real time PCR analysis of LCM endothelial tissue element from both skin and day 3 wound edge tissue, indicating that the expression of endothelial miR-199a-5p is down-regulated in response to wounding (Fig. 5B). The repression of miR-199a-5p was negatively associated with  Ets-1 serves as a bona fide miR-199a-5p target. A, in silico study revealing a possible binding site in Ets-1 3Ј-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. ␤-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 Ϯ S.E. ***, p Ͻ 0.001; *, p Ͻ 0.05 compared with corresponding control; ϩϩϩ, p Ͻ 0.001 compared with pLu-wild type Ets-1-3Ј-UTR plasmid transfected cells.

JOURNAL OF BIOLOGICAL CHEMISTRY 41037
DISCUSSION miR-199a-5p, first characterized in 2003, is derived from two genetic loci in human genome (chromosome 19 for miR-199a-1; chromosome 1 for miR-199a-2). It is well documented that inducible miR-199a-5p arrests cell proliferation and contributes to cell death (7,8,26,27). Our group and others have previously reported that miR-199a-5p regulates cellular detoxifying systems by target-ing multidrug resistance-associated protein 1 (6) and CD44 (12). In cardiovascular biology, although miR-199a-5p is known to play a critical role in regulating the function of cardiomyocytes (7, 8, 28 -31), information on its significance in vascular biology is scanty. This study provides the first evidence demonstrating that endogenous miR-199a-5p blocks angiogenic response by targeting master transcription factor Ets-1 in endothelial cells.
Ets-1 serves as a master transcription factor regulating angiogenic gene expression in endothelial cells including MMPs, urokinase type plasminogen activator, and vascular endothelial growth factor receptor 2 (3,25). Induction of Ets-1 expression has been reported during tissue repair such as skin burn wounds (40), gastric ulcer (41), and aortic injury (42) aiding angiogenesis. Pro-angiogenic stimuli induce the expression of Ets-1 via transcriptional control. Moderate hypoxia, a potent angiogenic stimulus, induces Ets-1 promoter activity and Ets-1 expression via activation of HIF (43). Hydrogen peroxide, a well known pro-angiogenic stimulus (44,45), activates nuclear factor (erythroid-derived 2)-like 2, leading to binding to antioxidant response elements and subsequent enhancement of Ets-1 promoter activity (46). Retinoic acid induces angiogenesis (47), with concomitant induction of Ets-1 transactivation (48). The findings from the current investigation, which agree with our previously published work (3), suggest an additional mecha-nism: Ets-1 expression is regulated through post-transcriptional modification in response to angiogenic stimulation. Indeed, miRs targeting Ets-1, namely miR-125b (49) and miR-222 (50), was also reported to serve as a potent angiostatic signal in endothelial cells (34,51,52), suggesting a sophisticated regulatory circuit in fine-tuning the behavior of endothelial cells in response to angiogenic signal.
In this study, we connect our in vitro observation to in vivo murine wound healing outcomes and report for the first time the involvement of miR-199a-5p-Ets-1 pathway in regulating MMP-1 expression in the context of cutaneous wound angiogenic response. Using the genetic approach, we demonstrate that the loss of Ets-1 compromises wound angiogenesis, compromises granulation tissue formation, and impairs wound closure. Intriguingly, mice with genetic deletion of Ets-1 show normal keratinocyte proliferation and differentiation (53), suggesting that the nonhealing phenotype of Ets-1 knock-out mice is likely independent of keratinocyte malfunction. We further characterized that MMP-1, which is reported to be vital in wound re-epithelialization (54), is specific to the miR-199a-5p-Ets-1 pathway. Consistent with findings from previous reports (25), Ets-1 regulates other MMP genes such as MMP-3 and MMP-9 in HMECs. However, the expression of these genes is not solely controlled by Ets-1. Instead, these genes are modulated by other transcription factors or transrepressors that might be targeted by miR-199a-5p, resulting in an off-set effect of the expression level.

miR-199a-5p Targets Ets-1
activation resulted in miR-199a-5p induction in human colon cancer cells (55). Interestingly, down-regulation of miR-199a-5p derepressed HIF-1␣ (7), resulting in a positive feed forward loop under modest low oxygen environment. Apart from the HIF-dependent pathway, Akt is another intracellular signal that is implicated in miR-199a-5p down-regulation. Insulin stimulation inhibits the expression of miR-199a-5p with the concomitance of Akt activation (8). Constitutively active Akt repressed miR-199a-5p expression and was associated with accumulation of miR-199a-5p target HIF-1␣ (8). Given the fact that activation of both HIF (56,57) and Akt (58,59) takes place in the early stage of skin wound healing, it is tempting to speculate that these two pathways might act simultaneously to suppress miR-199a-5p expression, resulting in derepression of Ets-1 and enabling wound angiogenesis in a MMP-1-dependent mechanism. Further investigation is required to charac-terize the upstream stimuli in regulating the wound-associated down-regulation of miR-199a-5p.
In summary, our results demonstrate a novel post-transcriptional control of Ets-1 expression by miR-199a-5p, and the regulation of its associated downstream mediator MMP-1. These findings reinforce the notion that miRs serve as common endogenous signals in regulating the motility of both epithelial and endothelial cells by targeting discrete set of genes. This work also provides the first evidence that wound-associated down-regulation of miR-199a-5p facilitates angiogenesis via desilencing Ets-1. Taken together, this investigation provides novel mechanistic insight explaining miR-dependent regulation of wound angiogenesis and provides the foundation of developing therapeutic intervention in treating complications of vasculopathy such as chronic nonhealing wounds.