MiR-191 inhibit angiogenesis after acute ischemic stroke targeting VEZF1

Acute ischemic stroke (AIS) is a major public health problem in China. Impaired angiogenesis plays crucial roles in the development of ischemic cerebral injury. Recent studies have identified that microRNAs (miRNAs) are important regulators of angiogenesis, but little is known the exact effects of angiogenesis-associated miRNAs in AIS. In the present study, we detected the expression levels of angiogenesis-associated miRNAs in AIS patients, middle cerebral artery occlusion (MCAO) rats, and oxygen-glucose deprivation/reoxygenation (OGD/R) human umbilical vein endothelial cells (HUVECs). MiR-191 was increased in the plasma of AIS patients, OGD/R HUVECs, and the plasma and brain of MCAO rats. Over-expression of miR-191 promoted apoptosis, but reduced the proliferation, migration, tube-forming and spheroid sprouting activity in HUVECs OGD/R model. Mechanically, vascular endothelial zinc finger 1 (VEZF1) was identified as the direct target of miR-191, and could be regulated by miR-191 at post-translational level. In vivo studies applying miR-191 antagomir demonstrated that inhibition of miR-191 reduced infarction volume in MCAO rats. In conclusion, our data reveal a novel role of miR-191 in promoting ischemic brain injury through inhibiting angiogenesis via targeting VEZF1. Therefore, miR-191 may serve as a biomarker or a therapeutic target for AIS.


INTRODUCTION
Acute ischemic stroke (AIS) is a major cerebrovascular disease ascribing to the sudden reduction of cerebral blood flow, characterized by a series of cellular and molecular disturbances. With a mortality rate of 10.3% and a morbidity rate of 19.7% in China [1], ischemic stroke is the leading cause of death and disability worldwide [2]. Several common risk factors, including hypertension, diabetes, dyslipidemia, alcohol, smoking, and inflammation [3][4][5], have been identified to be related to the pathogenesis of AIS. It is generally accepted that impaired neurovascular repair, especially angiogenesis, plays crucial roles in the development of ischemic cerebral injury [6].
Angiogenesis is the physiological process through which new blood vessels form by the extension or elaboration of existing vessels [7]. This process depending on endothelial cells is under the control of an extensive variety of angiogenic stimulators and inhibitors [8]. A growing number of studies have shown that microRNAs (miRNAs) are involved in the regulation of angiogenesis in ischemic diseases, including AIS [9][10][11].
Thus, the aim of the present study was to investigate the effects of angiogenesis-associated miRNAs in the angiogenesis after AIS both in vitro and in vivo.

Screening of miRNAs
To exclude tumor-related angiogenesis, we retrieved all published miRNAs related to the angiogenesis of endothelial cell in Pubmed (32 miRNAs, Table 1). After excluded 24 miRNAs that have been shown to play definite roles in acute stroke, we selected 8 miRNAs for further study ( Table 2).

Characteristics of enrollment patients
We enrolled 6 AIS patients and 6 control subjects as Cohort A, and another 12 AIS patients and 12 control subjects as Cohort B. Characteristics of Cohort A and B were shown in Table 3 and Table 4, respectively. There was no statistically difference of demographic or vascular risk factors between AIS patients and controls.

Expression of miR-191 in rat MCAO model and OGD/R HUVECs
Consistently, miR-191 levels were increased in the plasma of rat MCAO model both at 24h and 48h after reperfusion [14,34] (Figure 1E, 1F). However, no significant difference of miR-191 levels was observed between the two time points (data not shown). The expression of miR-191 was also increased in the ischemic boundary zone (IBZ) ( Figure 1G). We further detected miR-191 in HUVECs and found that miR-191 expression was elevated in OGD/R group ( Figure 1H).

Function of miR-191 in HUVECs proliferation
We transfected HUVECs with 50nM miR-191 mimic and 100nM miR-191 inhibitor to up and down-regulate the expression of miR-191, respectively (Figure 2A, 2B). HUVECs were then subjected to reoxygenation for 18h after 2h of OGD. We found that up-regulation of miR-191 significantly reduced HUVEC proliferation ( Figure 2C), while down-regulation of miR-191 promoted the proliferation ( Figure 2D).

Function of miR-191 in HUVECs apoptosis and cell cycle
By using flow cytometry, we showed that up-regulation of miR-191 increased the apoptosis rate of HUVECs ( Figure 3A, 3C), while down-regulation of miR-191 ameliorated the apoptosis induced by OGD/R ( Figure  3B, 3D). We also found that over-expression of miR-191 blocked the cell cycle in the S phase ( Figure 3E, 3G), which is consistent with the study of Gu, Y., et al. [35]. However, silence of miR-191 only slightly increased the number of G2 cells ( Figure 3F, 3H).

Function of miR-191 in HUVECs migration
To investigate the function of miR-191 in HUVECs migration in OGD/R, scratch wound healing assay and transwell migration assay were performed. Overexpression of miR-191 significantly delayed the closure of scratch wounds ( Figure 4A, 4C) and markedly reduced the number of migrated cells ( Figure 4E, 4G). In contrast, inhibition of miR-191 promoted the healing of scratch wounds ( Figure 4B, 4D) and enhanced cell migration ( Figure 4F, 4H).

Function of miR-191 in HUVECs tube-forming activity
We found that transfection of HUVECs with miR-191 mimic reduced the number of newly developed tube meshes when compared with controls ( Figure 5A, 5C).    In contrast, transfection of cells with miR-191 inhibitor promoted tube formation ( Figure 5B, 5D)

Function of miR-191 in HUVECs spheroid sprouting activity
We performed a 3-dimensional spheroid sprouting assay and demonstrated that over-expression of miR-191 significantly decreased the sprouting coverage area of HUVECs spheroids when compared with controls ( Figure 5E, 5G). In contrast, transfection of cells with miR191 inhibitor markedly enhanced the sprouting coverage area ( Figure 5F, 5H).

Validation of predictive target gene of miR-191
Vascular endothelial zinc finger 1 (VEZF1) is one of the target genes of miR-191 predicted by TargetScan 7.2 and miRDB ( Figure 6A). We found that VEZF1 mRNA levels were not influenced by miR-191 mimic or  AGING miR-191 interference ( Figure 6B). Since VEZF1 is a nucleus transcription factor, we then extracted nucleoprotein and found that over-expression of miR-191 decreased VEZF1 protein levels in nucleus, while miR-191 inhibitor increased the protein levels of VEZF1 ( Figure 6C, 6D). Further luciferase assay showed a significant decrease in the luciferase activity of wild-type VEZF1 3' UTR ( Figure 6E, 6F), indicating that VEZF1 is the target of miR-191.

Inhibition of miR-191 reduced infarction volume of MCAO rats
The rats randomly received an intracerebroventricular infusion of miR-191/NC antagomir or blank control 3 days prior to MCAO. Compared to the NC antagomir, miR-191 antagomir significantly reduced the miR-191 levels both in plasma and IBZ at 48 h after reperfusion in MCAO rats ( Figure 8A, 8B). We found that VEZF1 mRNA levels of IBZ were not influenced by miR-191 antagomir ( Figure 8C) which was consistent with the results of cell experiments. However, the protein levels of VEZF1 were increased significantly ( Figure 8D, 8E). We also found that rats receiving miR-191 antagomir had smaller brain infarct volumes than those with NC antagomir ( Figure 8F, 8G).

DISCUSSION
Impaired angiogenesis plays a crucial role in cerebral injury after acute ischemic attack [6]. MiRNAs have been shown to be important regulators involved in the AGING process of angiogenesis [10,40]. Here we showed for the first time that miR-191 was elevated in the plasma of AIS patients as well as in MCAO rat model and OGD/R HUVEC model. Over-expression of miR-191 promoted apoptosis but inhibited proliferation, migration, tube-forming and spheroid sprouting activity in HUVECs, while silence of miR-191 displayed opposite results. Mechanistically, we found that miR-191 directly regulated VEZF1, leading to the changes of a variety of angiogenesis-associated genes targeted by VEZF1. In vivo studies demonstrated that Inhibition of miR-191 could reduce the infarction volume induced by MCAO in rats. Therefore, miR-191 might be a novel therapeutic target for the treatment of AIS.
Angiogenesis is one of the key repair mechanisms for the ischemic injury induced by acute stroke [7]. Gu et al. [35] demonstrated that miR-191 was preferentially expressed in endothelial cells compared to other types of human cells and displayed antiangiogenic effect. In the present study, we performed a series of well-established angiogenesis assays and demonstrated that miR-191 is an inhibitor of angiogenesis with effects of suppressing proliferation, migration, tube formation and spheroid  AGING sprouting in HUVECs. Knockdown of miR-191 could reduce the infarction area induced by MCAO in rats and promote proliferation, migration, tube formation and spheroid sprouting in HUVEC. Our results complement nicely with a previous report showing that knockout of miR-191 reduced hepatic ischemiareperfusion injury through inhibiting inflammatory responses and cell death [41]. Another study also showed that up-regulated miR-191 participated in renal ischemia-reperfusion injury via inducing apop-tosis of renal tubular epithelial cells [42]. These results indicate that lowing miR-191 might be a potential therapy for ischemia-reperfusion injury. However, although we found that plasma levels of miR-191 were increased in patients with AIS, there might be false positive and negative results in the process of miRNA screening because of the relative small sample size.
Future studies with larger sample size will be needed to verify the exact roles of miR-191 in the diagnosis and prediction of AIS. AGING VEZF1 encodes a zinc finger transcription factor which is essential for developmental angiogenesis and lymphangiogenesis. Mammalian VEZF1 is expressed in the anterior-most mesoderm at E7.5 during development and is later restricted in the vascular endothelium [43]. VEZF1 knockout mice showed embryonic lethality caused by vascular remodeling defects and loss of vascular integrity, indicating that VEZF1 is a critical regulator of vascular development [44]. VEZF1 is thus proposed to act as a transcriptional activator of pro-angiogeneic genes including EDN1 [36], MMP2 [36], STMN1 [37], MMPs [38], CITED2 [39]. VEZF1 can be epigenetically regulated by histone acetylation and deacetylation [43]. VEZF1 is specifically expressed in endothelial cells and correlated with the differentiation and proliferation of endothelial cells in the embryonic vascular system [45]. A series of studies showed that VEZF1 could activate angiogenesis by promoting endothelial cells proliferation, migration and vessel network formation [37,39,46,47]. Our data demonstrated that VEZF1 is regulated by miR-191 at post-translational level. Luciferase reporter assay validated that VEZF1 is a direct target of miR-191. To further prove miR-191 inhibit HUVECs angiogenesis by targeting VEZF1, we measured the expression levels of the VEZF1 targets [36][37][38][39] after miR-191 interference. Our data shown that over-expression of miR-191 inhibited the expression of angiogenesisrelated genes including EDN1, MMP1, and STMN1. However, further intervention studies are needed to elucidate whether miR-191 inhibited angiogenesis via targeting VEZF1. In conclusion, our data reveal a novel role of miR-191 in promoting ischemic brain injury through inhibiting angiogenesis via targeting VEZF1, which in turn resulted in up-regulation of CITED2 and downregulation of MMP-1, STMN1. (Figure 9). MiR-191 might be served as a promising effective biomarker and therapeutic target for AIS.

Ethics statement
Investigation was conducted in accordance with the ethical standards and according to the Declaration of

Patient enrollment
Patients were recruited consecutively from the department of geriatrics of Sir Run Run Hospital, Nanjing Medical University from January to June 2018. There were total 18 patients with AIS and 18 controls. AIS patients were recruited from stroke center within six hours from the onset of the symptoms before thrombolytic therapy. AIS diagnosis was confirmed using clinical features and brain MRI by two investigators in a double-blinded manner according to the 2018 Guidelines for the Early Management of Patients With Acute Ischemic Stroke [2]. The control subjects were selected during the same period in the same hospital from the health examination center. Exclusion criteria including: under 18 or over 90 years old; history of intracranial hemorrhage; craniocerebral trauma; giant intracranial aneurysms; recent (within 3 months) history of intracranial surgery; malignant tumors. Informed consents were obtained from all subjects.

MCAO
Male Sprague Dawley rats (7-8w, 230 g-280 g) randomly divided into two groups (12 in each group) were obtained from Shanghai Sippr-BK laboratory animal Co. Ltd. Right MCAO was induced using an intraluminal filament as Longa, E.Z. et al [48] described. The body temperature was maintained at 37 °C with a homothermal blanket and physiological parameters were monitored during the surgical procedure. After anesthetized with 1 % pentobarbital sodium (0.5ml/100g), the right common carotid artery (CCA), external carotid artery (ECA) and internal AGING carotid artery (ICA) were sequentially isolated. An incision was made in the distal region of the CCA, then a 40mm long (diameter: 0.26-0.28mm) poly-L-lysine coated nylon monofilament (Beijing Shadong Biotechnology Co., Ltd.) was inserted into the ICA, and the monofilament was advanced approximately 18-20mm beyond the carotid bifurcation until mild resistance was encountered. The occlusion was sustained for 2 h. The sham group underwent similar procedures, but the monofilament was not advanced into the CCA.
Neurological evaluations were performed according to an established graded scoring system at 24 h after reperfusion to verify the modeling success in function [49]. Briefly, neurological deficits were scored as follows: 0, no deficit; 1, failure to extend left forepaw upon lifting the whole animal by tail; 2, grip strength weakening of the left forepaw; 3, circling to the left when held by the tail; and 4, spontaneous circling.
Peripheral venous blood was collected at 24 and 48 hours after reperfusion and the brains were removed and frozen at −20 °C for 15 min at 48 hours after reperfusion when sacrificed (Supplementary Figure 3). To histologically verify the success of the model, coronal sections were cut into 2 mm thick slices, stained with 1% 2,3,5-triphenyltetrazolium chloride (TTC) at 37 °C in the dark for 20 min, and photographed (Supplementary Figure 4).

Plasma and brain fragments collection and storage
Peripheral blood samples were collected in tubes anticoagulated with ethylenediamine tetraacetic acid (EDTA) dipotassium salt after a definite diagnosis before any medication is administered. The samples were centrifuged (1000×g, 5minutes, 4 °C, Beckman Coulter) to remove blood cells and debris, and then transferred to 1.5ml microtubes (Axygen, MCT-150-C) for storage at -80°C until further processing. Brain fragments were collected and stored in liquid nitrogen.

Primary HUVECs isolation and culture
Fresh umbilical cords were obtained following delivery of healthy babies to healthy mothers. HUVECs were isolated from umbilical cords according to a previously described method [50]. Briefly, the umbilical vein was inserted an intravenous needle for single use with tube and flushed with 30ml 37 °C phosphate buffer saline (PBS), following which the cord was clamped at the distal end and the vessel filled with collagenase (Type Ia, 1 mg/mL, Sigma, C9891), until mildly distended. Following incubation at 37°C for 10-15 min, the cord was unclamped and the digest drained. The vessel was gently massaged and flushed through with 20ml endothelial cell medium (ECM, Science, 1001), and the digests were pooled. The endothelial cell suspension was centrifuged (500×g, 5 min), and the cell pellet was resuspended in ECM. This suspension was seeded into a 25ml flask (Corning, 430639).

OGD/R
The OGD/R protocol was performed to mimic ischemia in vitro. Briefly, culture medium was removed and rinsed with PBS for three times. HUVECs were placed into a tri-gas incubator (memmert, Eastern Friesland, Germany) containing 1% O2, 5% CO2, 94% N2 at 37 °C with glucose-free Dulbecco's Modified Eagle Medium (DMEM, Gibco, 11966025, US). After two hours challenge, DMEM was replaced with ECM. The cells were maintained for further 24 h at 37 °C in a humidified 5% CO2 incubator to generate reperfusion.

Total RNA isolation
Total RNA was isolated from plasma, brain and cell samples using TRIzon (CWBio, CW0580) reagent following the manufacturer's instructions. RNA concentration and purity were determined with one drop spectrophotometer (OD-1000+, Nanjing wuyi Science and Technology Co., Ltd., China).

Quantitative real-time polymerase chain reaction (qRT-PCR)
CDNA was generated from 1 µg RNA using miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (vazyme, MR101-02, Nanjing, China) for miRNA or PrimeScript™ RT reagent Kit (Perfect Real Time) (Takara, RR047A, Japan) for mRNA. Real-time PCR was performed using QuantStudio 5 (Applied Biosystems, US) with miRNA Universal SYBR qPCR Master Mix (vazyme, MQ101-02) for miRNA or Maxima SYBR Green/ROX qPCR Master Mix (2X) (Thermo Scientific™, K0221, US) for mRNA according to the manufacturer's protocol. All reactions were run in triplicate and relative gene expression was calculated using the comparative threshold cycle (Ct) method (relative gene expression = 2− (ΔCtsample-ΔCtcontrol) ). The U6 snRNA was used as an internal control for miRNA while TATA-binding protein (TBP) used for mRNA. The genespecific primers sequences are listed in Table 5.

Cell proliferation assay
To assess the proliferation rate of HUVECs, Cell Counting Kit-8(CCK-8) assays (Dojindo, Japan) were performed according to the manufacturer's instructions.
Briefly, 3000 HUVECs in 100 μl of cell suspension were seeded in 96-well flat-bottomed plates. After 8 hours incubation, CCK-8 reagent was added to each well, and the absorbance of each well was measured at 450 nm after 3 hours incubation by a microplate reader (Synergy H1, BioTek, US). A value of 100% was assigned to the respective control group.

Cell migration assay
Scratch wound healing assay and transwell migration assay were performed to evaluate the motility of HUVECs. For the scratch wound healing assay, 5×10 5 of HUVECs were seeded in a 6-well plate. After reaching confluence, the cell monolayer was scratched with a pipette tip (10 μl) to generate 4 scratch wounds and then rinsed twice with PBS to remove nonadherent cells. Phase-contrast light micrographs were taken immediately after scratching (0 h) as well as after 6h and 12 h with ×200 magnification using a CKX41 microscope (Olympus, Japan).
For the transwell migration assay, 2×10 5 of HUVECs in 500μl 1% FBS ECM were seeded into a 24-well insert (costar, 3422), and 750μl of 15% FBS ECM was added to the lowerwell. After 24h of incubation, nonmigrated cells were removed with cotton swabs, and migrated cells were stained with 0.1% crystal violet (Solarbio, C8470, Beijing, China). The number of migrated cells was determined in 3 microscopic regions of interest at ×200 magnification using a CKX41 microscope.

Tube formation assay
To

Spheroid sprouting assay
HUVECs were suspended in ECM containing 0.25% (w/v) methylcellulose (Sigma-Aldrich) and seeded (1000 cells/100μl) in low attachment, round-bottom, 96-well spheroid microplates (Corning, 4520). After incubation for 24 h, spheroids were harvested and resuspended in 20μl Matrigel. The spheroid-containing Matrigel was rapidly transferred to 24-well plates and allowed to polymerize for 30 min, after which 500μl ECM was added to each well. After 24h of incubation, the spheroid-sprouting capacity was quantified by measuring the sprouting coverage area of the sprouts using imageJ software.

Protein extraction and Western blots
Nuclear extracts were prepared with NE-PER™ Nuclear and Cytoplasmic Extraction Reagents according to the AGING manufacturer's protocol (Thermo Scientific, 78833, US). Protein concentrations were measured by BCA protein assay (Beyotime Biotechnology, Shanghai, China). Equal amounts (20 μg) of protein were separated by 4-20% GenScript SurePAGE, Bis-Tris, precast polyacrylamide gels (GenScript Biotechnology, Nanjing, China) electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Membranes were then incubated overnight at 4 °C with a 1:1000 dilution of anti-Histone H3 (Cell Signaling Technology, 4499s, US) and anti-VEZF1 (Proteintech, 19003-1-AP, US). After additional incubation with a 1:5000 dilution of anti-rabbit IgG (heavy and light chain) antibody (CST, 7074S) for 2 h, the immune complexes were detected by Immobilon Western HRP Substrate Peroxide Solution (Millipore Corporation, Billerica, MA 01821, USA). And images were acquired using ChemiDoc TM XRS+ Imaging System (Bio-rad, US). The intensity of immunoreactivity was assessed using Image Lab 6.0 software.

Intracerebroventricular injection of the miR-191 antagomir
The miR-191 antagomir and NC antagomir were purchased from RiboBio(Guangzhou, China). The NC and miR-191 antagomir (2.5 μg/2.5 μl) were diluted with 1.25 μl of Entranster TM in vivo transfection reagent (Engreen,18668-11-1, Beijing, China). The solution was mixed with 1.25μl PBS gently, kept at room temperature for 5 min and then injected intra-cerebroventricularly (i.c.v.) using a microsyringe (KD Scientific Inc., USA) under the guidance of a stereotaxic instrument (RWD Life Science). A solution of 3.75μl PBS added with 1.25 μl of Entranster TM in vivo transfection reagent was acted as blank control. Intracerebroventricular injection was performed according to a previously described method [51]. The stereotaxic coordinates the right lateral ventricle: ML: -1.40mm, AP: -0.36mm, DV: -3.90mm.

Statistics
Differences between the two groups were analyzed by the unpaired Student's t test or Mann-Whitney test after testing the distribution of the data. Differences between multiple groups were analyzed by one-ANOVA followed by the Student-Newman-Keuls post hoc test (Graphpad Prism 7.0, USA) after testing the data for equal variance. All values were expressed as mean ± SEM. Statistical significance was accepted at P<0.05.

AUTHOR CONTRIBUTIONS
Wei Gao and Xiang Lu designed the research, interpreted the data, and contributed to revising the manuscript. Kang Du and Can Zhao performed the research, analyzed the data, and wrote the manuscript. Li Wang and Yue Wang contributed to data collection and performance of rats MCAO model. Kangzhen Zhang, Xiyu Shen and Huixian Sun contributed to recruitment of patients and clinical diagnosis of disease.