MiR‐205 promotes endothelial progenitor cell angiogenesis and deep vein thrombosis recanalization and resolution by targeting PTEN to regulate Akt/autophagy pathway and MMP2 expression

Abstract MicroRNAs (MiRNAs, MiRs) represent a class of conserved small non‐coding RNAs that affect post‐transcriptional gene regulation and play a vital role in angiogenesis, proliferation, apoptosis, migration and invasion. They are essential for a wide range of physiological and pathological processes, especially for vascular diseases. However, data concerning miRNAs in endothelial progenitor cells (EPCs) and deep vein thrombosis (DVT) remain incomplete. We explored miRNAs that modulate angiogenesis in EPCs and thrombolysis, and analysed their underlying mechanisms using a DVT model, dual‐luciferase reporter assay, qRT‐PCR, Western blot, immunofluorescence staining, flow cytometry analysis, CCK‐8 assay, angiogenesis assay, wound healing and Transwell assay. We found that miR‐205 enhanced the homing ability of EPCs to DVT sites and promoted thrombosis resolution and recanalization, which significantly reduced venous thrombus. Additionally, we demonstrated that miR‐205 overexpression significantly enhanced angiogenesis in vivo and in vitro, migration, invasion, F‐actin filaments and proliferation in EPCs, and inhibited cell apoptosis. Conversely, down‐regulation of miR‐205 played the opposite role in EPCs. Importantly, this study demonstrated that miR‐205 directly targeted PTEN to modulate the Akt/autophagy pathway and MMP2 expression, subsequently playing a key role in EPC function and DVT recanalization and resolution. These results elucidated the pro‐angiogenesis effects of miR‐205 in EPCs and established it as a potential target for DVT treatment.


| INTRODUC TI ON
Deep vein thrombosis (DVT) refers to the formation of blood clots in deep venous lumen, causing blood flow disorders, and its prevalence increases with age. 1,2 DVT with its major complications, post-thrombotic syndrome (PTS) and pulmonary embolism (PE) can be lifethreatening, which is a global health problem. [3][4][5] PTS consists of pain, swelling and even ulceration of the leg that can occur immediately after DVT or can delay the onset. [6][7][8] Prophylaxis and treatments for DVT patients include anticoagulation by heparin, vitamin K antagonists, and thrombin and Factor Xa inhibitors. Deep vein thrombosis surgical intervention consists of thrombectomy or catheter-based thrombolysis using urokinase, streptokinase or tissue plasminogen activator. 9 Unfortunately, these methods are not always effective.
Studies have shown that the main consequences of DVT are death, recurrence, PTS and major bleeding caused by anticoagulation. 4,10 Thus, it is essential to study the molecular mechanism of DVT and explore alternative therapies for DVT treatment.
Of note, angiogenesis and thrombolysis are crucial for thrombus recanalization. [11][12][13] Numerous studies have shown that endothelial progenitor cells (EPCs) are recruited to the thrombus to accelerate thrombus resolution and play a vital role in physiological and pathological neovascularization in adults. 12,[14][15][16] Moreover, EPCs can differentiate into endothelial cells to participate in angiogenesis and thus are a promising therapeutic approach for thrombus resolution as there is only limited success with the current treatment options. 12,16,17 However, the function of EPCs can be impaired by cardiovascular risk factors, ischaemic disease and graft vasculopathy. [18][19][20][21] Therefore, it is important to explore methods to improve EPC homing and angiogenesis.
MicroRNAs (MiRNAs, MiRs) are small non-coding RNAs consisting of approximately 22 nucleotides that influence posttranscriptional gene regulation by targeting the 3′ untranslated regions (3′UTRs) of mRNAs. [22][23][24][25] They play a major role in the regulation of cell migration, proliferation, apoptosis and angiogenesis, which is essential for the development and progression of vascular disease. 26 Our previous studies have suggested that miR-NAs including miR-483-3p, miR-150, let-7e-5p and miR-126 play a crucial role in regulating angiogenesis and migration in EPCs. [27][28][29][30] A recent study has also indicated that miR-126-loaded EPC-derived exosomes are a promising potential vehicle for DVT therapy. 11 Among the emerging miRNAs, miR-205 has an important impact on the progression of diseases, but it is a "double-edged sword" in diseases. Studies have shown that miR-205 can facilitate wound healing in human corneal epithelial cells, 31 while it promotes tumour angiogenesis and cancer cell proliferation and migration in human nasopharyngeal carcinoma, lung cancer, breast cancer and endometrial cancer. [32][33][34][35] On the contrary, miR-205 has been shown to play an antiproliferation role in certain tumours. 36 MiRNAs can cooperatively target a variety of mRNAs, and the same mRNA can be regulated by multiple miRNAs, and therefore, the role of miRNA may vary with cellular content and stimulation, which provides a possible explanation for the complex and even controversial functions of miR-205. Recently, it has also been confirmed that miR-205 promotes ovarian cancer cell invasion by enhancing the expression of matrix metalloproteinase (MMP) 2/9. 37 MMP2 is an essential factor for promoting EPC migration and early venous thrombus resolution. [38][39][40] However, the role and underlying mechanism of miR-205 in DVT and EPCs, particularly in regulating angiogenesis and thrombus resolution, remain largely unknown.
Here, we showed the role of miR-205 in EPCs and DVT recanalization and resolution, and explored the underlying molecular mechanism, which is crucial in the pathogenesis of DVT and contributes to the development of a novel effective treatment for DVT.

| Cell culture and transfection
Endothelial progenitor cells were cultured in endothelial basal medium-2 (EBM-2; Lonza) and EGM-2 MV SingleQuot Kit Suppl. & Growth Factors (Lonza) and maintained at 37°C in a 5% CO 2 incubator. Lentiviral particles carrying vehicle control (NC), miR-205 mimics and inhibitor were provided by GenePharma. They were used to infect EPCs according to the manufacturer's instructions.

| Quantitative real-time PCR analysis
Total RNA was extracted by TRIzol reagent (Invitrogen). The PrimeScript RT reagent kit (TaKaRa) was used for first strand of cDNA synthesis, and the SYBR Green quantitative PCR Master Mix (Bio-Rad) was used for gene amplification. MiRNA levels were determined following the instructions of the Hairpin-it™ miRNAs RT-PCR Quantitation Kit (GenePharma). MRNA expression levels were analysed using mRNA primers (GENEWIZ). The endogenous control for miR-205 was U6 RNA, and for the other transcripts, it was GAPDH. Quantitative real-time PCR (qRT-PCR) was performed on a High-Throughput Quantitative PCR LightCycler480 II (Roche Applied Science).

| In vivo angiogenesis assay
Male nude mice (5 weeks old) were provided by Shanghai Lingchang Biotechnology Co., Ltd. and raised in the Experimental Animal Images were obtained by an Olympus DP73 microscope.

| In vitro angiogenesis assay
Matrigel (50 μL/well) was transferred to a 96-well plate, followed by inoculation of transfected EPCs (3 × 10 4 cells). After 12 hours, images were captured with an inverted microscope. The extent of tube formation was assessed by measuring the tube number using Image J software.

| Analysis of the therapeutic effect on deep vein thrombosis
Male nude mice (10 weeks old) were anesthetized, and the left jugular vein was isolated under a stereo microscope, and the proximal end of the heart and its branches were ligated. GFP-miR-205 EPCs and GFP-NC EPCs (5 × 10 6 ) were injected into the nude mice through the orbital vein at 48 hours after DVT. After 7 days, thrombi with vessel walls were harvested for measurements of size and weight, and for immunofluorescence. The number of GFP-EPCs at the thrombus was observed under an IX-81 laser confocal microscope (Olympus) to analyse homing ability.

| Migration assay
Endothelial progenitor cells were seeded in 6-well plates (3 × 10 5 cells/ well), and a 200-µL pipette tip was used to create a linear scratch at nearly 95% confluence. Subsequently, fresh EBM-2 medium without FBS was added to prevent EPC proliferation. Images were captured by an inverted microscope at 0 and 12 hours, and the number of migration cells was assessed by Image J and GraphPad Prism software.

| Invasion assay
Corning Transwell chamber in 24-well plates was used to perform cell invasion assays. Matrigel was added to the upper chamber and placed at 37°C for 30 minutes, followed by the addition of cells. EBM-2 medium supplemented with 10% FBS was transferred to the lower compartment of the chamber. Images of invasion cells were obtained using an inverted microscope. The invasion assay analysis was performed using Image J software and GraphPad Prism software.

| Cell proliferation and apoptosis assays
Cell Counting Kit-8 (CCK-8, Dojindo) was used for cell proliferation assay according to the specification. Briefly, transfected EPCs were seeded in 96-well plates (3 × 10 3 cells/well) and examined at 0, 24, 48, 72 and 96 hours. The detection buffer (100 μL, ratio of medium to CCK-8 was 9:1) was added to each well at different time-points.
After incubation for 2 hours at 37°C, the absorbance at 450 nm was detected by Multi-Detection Readera (SpectraMax5). For cell apoptosis analysis, the APC Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend) was used according to the instructions. All the tests were performed at least three times.

| Western blot analysis
Total cellular protein was lysed in RIPA buffer supplemented with protease inhibitors, phosphatase inhibitors and PMSF, and centrifuged at 12 000 g for 10 minutes at 4°C. The BCA Protein Assay Kit (Beyotime, P0011) was used to quantify protein levels. Cellular proteins were separated by 10% or 15% SDS-PAGE gels and transferred to polyvinylidene IRDye® 800CW goat anti-rabbit or antimouse IgG antibodies (LI-COR) were used at 1:10 000 dilution. The immune bonds were detected using an Infrared Imaging System (LI-COR).

| Statistical analysis
All data are presented as mean ± SEM. Student's t test and oneway ANOVA were used to analyse significant differences between groups. GraphPad Prism software was used for statistical analysis. P < .05 was considered statistically significant.

| MiR-205 promotes EPC homing to the thrombus, and deep vein thrombosis recanalization and resolution
Endothelial progenitor cells exert an important role in DVT mainly by regulating angiogenesis and thrombolysis. 13 However, the function of EPCs can be impaired by a poor microenvironment in the body.
In this study, a model of DVT was established by ligation of a jugular vein and its branches. Endothelial progenitor cells infected with NC or miR-205 mimics were intravenously administrated into nude mice. The results showed that the thrombus size and weight were significantly reduced on day 7 after injection of GFP-miR-205 EPCs compared with GFP-NC EPCs (P < .01, Figure 1A

| MiR-205 promotes EPC angiogenesis in vivo and in vitro
The role of EPCs in DVT recanalization and resolution is associated with their migration, invasion and angiogenesis abilities. In the miR-205 overexpression group, angiogenesis in the thrombus was  Figure 2C). The results suggested that the tube formation of EPCs in miR-205 mimics group was significantly increased, while angiogenesis in EPCs of miR-205 inhibitor group was attenuated, and there were no differences between the N group and NC group (P > .05, Figure 2D). These results suggested that miR-205 played a vital role in EPC-associated angiogenesis.

| MiR-205 enhances EPC migration and invasion by regulating F-actin and MMP2
To investigate effect of miR-205 on EPC migration and invasion, wound healing and Transwell invasion assays were performed.
Wound healing assay revealed that the ability of EPC migration in the miR-205 mimics group was much higher than that in the NC group (P < .01), and the EPC migration ability of the miR-205 inhibitor group was significantly reduced than that of the NC group (P < .01, Figure 3A,B). However, no significant difference was observed between untransfected and negative control cells (P > .05). Analogously, invasion assay shared similar results, whereby miR-205 mimics increased cell invasion ability significantly (P < .001), while miR-205 inhibitor obviously impaired the ability of cell invasion (P < .01), with no significant difference between N group and NC group (P > .05, Figure 3C,D). Further, F-actin and MMP2, which are closely related to cell migration ability, were observed. As shown in Figure 3E Figure 3F-H). These results disclosed that miR-205 played a key regulatory role in EPC migration and invasion, at least in part, by regulating F-actin filaments and MMP2 expression.

| PTEN directly binds to miR-205
Based on the miRNA database (http://www.mirba se.org), targetScan, RNA hybrid software and using bioinformatics methods to predict miR-205 targets and analyse its functions, PTEN was found to be associated with miR-205 and cell growth. Importantly, we demonstrated that the sequence of miR-205 is complementary to the target sequence in the 3′UTR of PTEN and that their hybridization was predicted by RNA hybrid software, requiring a minimum free energy of -28.6 kcal/mol ( Figure 5A). To further explore whether miR-205 binds to the PTEN 3′UTR, the luciferase reporter assay was performed. As shown in Figure 5B, wild-type (WT) PTEN and mutant (MU) PTEN were inserted into a luciferase reporter plasmid cleaved with HindIII and SpeI (TaKaRa) endonucleases. The dual-luciferase reporter assay showed that only the group cotransfected with the F I G U R E 2 Effects of miR-205 on angiogenesis in vivo and in vitro. A, In vivo angiogenesis was evaluated at Day 7 after subcutaneous injection of Matrigel-mixed EPCs into nude mice. B, Haematoxylin and eosin (H&E) staining: miR-205 overexpression and knockdown, respectively, increased and decreased tube formation by EPCs in vivo (magnification, ×200). C, Effects of miR-205 on tube formation in EPCs (magnification, ×100). D, MiR-205 overexpression and knockdown, respectively, increased and decreased tube number. ***P < .001 compared with NC group WT PTEN plasmid and the miR-205 mimics had decreased luciferase activity, while the other groups did not change significantly ( Figure 5C), confirming that miR-205 interacts with the 3′UTR of PTEN and that the binding region is consistent with the prediction.
Furthermore, qRT-PCR and Western blot analyses suggested that the PTEN mRNA and protein expression levels were reduced in the miR-205 mimics group, whereas they were increased in the miR-205 inhibitor group, compared with the NC group ( Figure 5D-F). Taken together, we concluded that PTEN was a direct target of miR-205 in the regulation of EPCs.

| Effects of cotransfection of miR-205 and PTEN on tube formation, cell migration and invasion of EPCs
To test the effects of cotransfection of miR-205 mimics and PTEN on EPC function, tube formation, migration and invasion assay were performed. The tube formation experiment demonstrated that miR-205 mimics markedly enhanced tube formation ability, and PTEN significantly suppressed tube formation in EPCs, compared with the NC group. Furthermore, the tube formation capacity of EPCs in the miR-205 mimics + PTEN cotransfection group F I G U R E 3 Effects of miR-205 on cell migration and invasion in EPCs. A, Wound healing assay showing the effects of miR-205 on EPC migration. Scale bar = 100 μm. MiR-205 overexpression and knockdown, respectively, decreased and increased the cell number of EPC migrated. B, Effects of miR-205 on cell migration ability were analysed. **P < .01 compared to the NC group. C, Transwell cell invasion assay provided results similar to those for wound healing. Scale bar = 100 μm. D, Effects of miR-205 on cell invasion ability were analysed. **P < .01, ***P < .001 compared with NC group. E, Effects of miR-205 on F-actin in cultured EPCs. Cells were fixed, permeabilized, stained with rhodamine-phalloidin and DAPI, and visualized by confocal microscopy; down-regulation of miR-205 impaired F-actin filaments, while miR-205 overexpression prevented disruption of F-actin filaments. F-H, MMP2 mRNA and protein expression levels were observed by qRT-PCR and Western blot analysis. *P < .05, **P < .01 compared with NC group decreased and increased compared with the miR-205 mimics group and the PTEN group, respectively. However, there was no significant difference in tube formation of EPCs between the miR-205 mimics + PTEN cotransfection group and the NC group (P > .05; Figure 6A,B). The migration experiment demonstrated that the number of migration EPCs in the PTEN group was markedly decreased, whereas in the miR-205 mimics group, it was obviously increased than in the NC group ( Figure 6C,D). Analogously, the Transwell invasion assay showed similar results, that is miR-205 overexpression significantly promoted cell invasion, while PTEN overexpression inhibited cell invasion ( Figure 5E,F). The results confirmed that miR-205 targeted PTEN to modulate EPC angiogenesis, migration and invasion.

| Effects of cotransfection of miR-205 and PTEN on cell autophagy and MMP2 expression in EPCs
It has been shown that PTEN is a negative regulator of the PI3K/Akt pathway. 41 The Akt pathway is closely related to cell autophagy and MMP2 expression. 21 In addition, venous thrombus resolution and

| D ISCUSS I ON
In the present study, we identified the role of miR-205 in DVT recanalization and resolution, and revealed its regulatory functions and molecular mechanisms in EPCs ( Figure 7G [32][33][34][35]42 Meanwhile, miR-205 promotes wound healing in human corneal epithelial cells. 31 However, the role and underlying mechanism of miR-205 in DVT and EPCs, particularly in regulating angiogenesis and thrombus resolution, remain to be elucidated.
Studies have shown that blood vessels are rich in stem/progenitor cells expressing the surface marker CD34, a marker for evaluating angiogenesis. [43][44][45] Moreover, MMPs play a crucial role in degradation of the extracellular matrix and basement membrane components to allow the migration and invasion of leucocytes and other cell types in vascular diseases and homeostasis. 39,46 In particular, MMP2 is an essential factor for promoting early venous thrombus resolution. 38,39 Therefore, the expression of CD34 and MMP2 in the venous thrombus was examined by immunofluorescence.
Here, we showed that miR-205 overexpression in EPCs significantly reduced thrombus size and weight, and enhanced EPC homing and the expression of CD34 and MMP2. Therefore, we speculated that miR-205 may promote DVT recanalization and resolution, at least in part, via enhancing angiogenesis and migration in EPCs.
To test our hypothesis, in vivo angiogenesis experiments, cell function experiments and molecular biology experiments were carried out. Our results showed that miR-205 overexpression significantly promoted angiogenesis in vivo and in vitro, migration and invasion in EPCs. Cell migration has been shown to be strongly connected to attachment of the leading edge, cellular contraction by myosin inserting between F-actin filament bundles and detachment of the adhesions at the trailing cell rear, which requires F-actin for each step of the process. 47 Additionally, MMP2 is a key regulator of EPC migration. 40 To detect whether miR-205 influences EPC migration by regulating F-actin filaments and MMP2, we performed F I G U R E 6 Effects of cotransfection of miR-205 and PTEN on tube formation, cell migration and invasion of EPCs. A-B, In vitro tube formation ability was analysed (magnification, ×100). C-D, The migration ability of EPCs was detected by wound healing assay. Scale bar = 20 μm. E-F, The number of invasion cells was observed by Transwell invasion assay. Scale bar = 100 μm. **P < .01, ***P < .001 immunofluorescence of F-actin, and qRT-PCR and Western blot of MMP2. As shown in Figure 3E way. 48,49 Furthermore, PTEN has been reported to play a major role in inhibiting migration and invasion of tumour cell via the Akt/SP1/ MMP2 pathway. 50 Intriguingly, autophagy inhibition promoted EPC migration by up-regulating MMP2 expression. 21,40 In addition, it has been verified that miR-205 enhanced ovarian cancer cell invasion by F I G U R E 7 Involvement of the autophagy pathway in miR-205-mediated effects in EPCs on MMP-2. A-F, Western blot analysis of the expression of PTEN, Akt, p-Akt, LC3-II, p62 and MMP2 in EPCs after transfected with NC and miR-205 mimics or cotransfected with miR-205 mimics and PTEN, and PTEN overexpression plasmid. MiR-205 overexpression up-regulated the expression of Akt phosphorylation, p62 and MMP2, and inhibited PTEN and LC3 II in EPCs compared with NC group, while cotransfection of miR-205 mimics and PTEN in EPCs could effectively antagonize miR-205-induced autophagy suppression and the expression of p-Akt and MMP2, and overexpression of PTEN reduced the expression levels of Akt phosphorylation, p62 and MMP2, and increased PTEN and LC3 II in EPCs. *P < .05, **P < .01 compared with NC group; #P < .05, ##P < .01 compared with miR-205 mimics + PTEN group. G, Principle signalling pathways of miR-205 up-regulation involved in promoting DVT recanalization and resolution, and EPC angiogenesis. MiR-205 enhanced angiogenesis, migration and invasion in EPCs by directly targeting PTEN, regulating MMP-2 mRNA and protein expression via Akt/autophagy pathway in EPCs. Additionally, F-actin filaments were involved in the migration ability of miR-205 in EPC regulating MMP2/9 expression in vitro. 37 Thus, we examined the effects of miR-205 overexpression, co-expression of miR-205 and PTEN, and PTEN overexpression on PTEN/Akt/autophagy pathway and MMP2 expression in EPCs. Our results showed that coexpression of miR-205 and PTEN in EPCs effectively antagonized the miR-205-induced autophagy suppression, MMP2 expression and EPC functions. Thus, this study revealed that miR-205 targeted PTEN, to regulate MMP2 expression via Akt/autophagy pathway, thereby mediating angiogenesis, cell growth and migration of EPCs, subsequently enhancing the ability of EPC to homing to the thrombus to accelerate venous thrombus recanalization and resolution.
In summary, we demonstrate that miR-205 directly targets PTEN to modulate MMP2 expression via the Akt/autophagy pathway, thus playing a key role in EPC function and DVT recanalization and resolution. This study provides valuable information for exploring miR-NAs in the treatment of DVT.