Introduction
Deep vein thrombosis (DVT) is a common manifestation of venous thromboembolism (VTE) and is defined as a form of thrombophlebitis associated with the formation of blood clots in deep veins. DVT is sometimes asymptomatic but often presents as non-specific symptoms, such as discomfort or pain in the legs, or a feeling of fever. Pain, tenderness, swelling or blue or red discoloration of the limbs are the typical symptoms [
1]. Oral anticoagulants are the first-line treatment for VTE to hasten thrombus resolution because of lower bleeding risk. It takes at least 3 months to take anticoagulants to prevent early recurrence [
2]. Endothelial progenitor cells (EPCs) could modify endothelial regeneration, revascularization, vascular activity and angiogenic factor secretion, protease production, thrombosis and recurrence prevention, and vein wall remodeling [
3]. In addition, EPCs can form new blood vessels by differentiating into endothelial cells, which could be utilized as a promising therapeutic regimen for DVT-associated thrombus resolution in patients achieved limited success [
4]. Therefore, targeting EPCs is a promising direction for the resolution of DVT.
Several long noncoding RNAs (lncRNAs) are listed as essential partners in multitudinous physiological and pathological processes of DVT, including proliferation, migration, and angiogenesis of EPCs [
4,
5]. Specifically, lncRNA taurine upregulated gene 1 (TUG1) is a unique modifier for EPC function and angiogenesis under diabetic conditions [
6]. Moreover, a report on aneurysms proposes that TUG1 overexpression induces EPC migration, invasion, and differentiation [
7]. But, little is known about TUG1-mediated influence on EPCs angiogenesis in the setting of DVT. According to the latest annotation by the Ensembl database, there are 8 transcripts (isoforms) of Tug1 (
https://www.ensembl.org/Mus_musculus/Gene/Summary?db=core;g=ENSMUSG00000056579;r=11:3589785-3599673). Tug1–202 is a transcript with length ≥ 200 bp and number of exons ≥2. After calculation, the minimum coverage of TUg1–202reads is ≥3, so subtype Tug1–202 was selected for our study. LncRNAs-mediated competitive binding of microRNAs (miRs) plays a vital role in regulating RNA transcription. It has been widely explored that various miRs involve in the pathological possesses of DVT, including vascular endothelial cell physiology [
8], autophagy and tube formation of EPCs [
9], and recanalization and resolution [
10]. It has been implicated that miR-92 could modify vascular smooth muscle cell function in vascular restenosis and injury [
11]. miR-92a-3p is associated with oxidative stress in central venous catheter-related thrombosis (CRT) [
12], and miR-92a-3p inhibition could hasten angiogenesis of endothelial cells, serving as a potential target for the treatment of atherosclerosis [
13]. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (Hmgcr) is the rate-limiting enzyme for the biosynthesis of cholesterol and isoprenoids. Jayoung Choi et al. have specified that mevalonate, a metabolic product of Hmgcr, could restore venous angiogenesis [
14].
Based on former publications, we studied TUG1-mediated EPCs angiogenesis and DVT resolution via miR-92a-3p/Hmgcr axis, expecting to discover a molecular way for the disease management.
Materials and methods
Ethical approval
This research was processed with the approval of the animal ethic committee of Beijing Haidian Maternal & Child Health Hospital.
Isolation and culture of EPCs
Using Histopaque 1077 density gradient centrifugation (Sigma-Aldrich, MO, USA), bone marrow mononuclear cells (MNCs) were isolated from C57 mice. MNCs/cm
2 (10
6) were seeded on fibronectin-coated six-well plates in endothelial cell growth medium-2MV (EGM-2MV, Lonza, MD, USA) containing 10% fetal bovine serum, 1% streptomycin and penicillin, and then cultivated in a constant temperature incubator at 37 °C with 5% CO
2. The adherent cells were removed 72 h later. Since that, the medium was renewed every 3 days [
15].
After 10 d, adherent cells were fixed with 2.5 mg/mL DiI-acetylated-low density lipoprotein (DiI-ac-LDL, Peking Union-Biology, Beijing, China) for 2 h and with 2% paraformaldehyde (Sigma) for 5 min. Subsequently, cells were incubated with 10 mg/L fluorescein isothiocyanate labeled ulex europaeus agglutinin (Sigma) for 1 h.
EPCs were incubated with primary antibodies CD133 and FLK-1 (both from Abcam, Cambridge, UK) and combined with Cy3 (BOSTER, Wuhan, China) or fluorescein isothiocyanate (FITC; Santa Cruz, CA, USA). Representative micrographs were obtained with a microscope (Olympus, Tokyo, Japan) [
16].
EPCs transfection
Lipofectamine 3000 (Invitrogen, CA, USA) was utilized for EPCs transfection. The transfection plans included oe-NC, oe-TUG1, NC inhibitor, miR-92a-3p inhibitor, NC siRNA, Hmgcr siRNA, oe-TUG1 + NC mimic, oe-TUG1 + miR-92a-3p mimic, miR-92a-3p inhibitor + NC siRNA and miR-92a-3p inhibitor + Hmgcr siRNA (Antpedia, Shanghai, China). Cells were collected after 48 h of transfection.
Growth factor-reduced Matrigel (BD Biosciences, NJ, USA) was solidified in 96-well plates at 50 μL/well. EPCs (1 × 10
4) were resuspended in 200 μL endothelial basal medium-2 without EGM-2 SingleQuots, and added onto Matrigel. Tube images were captured after 18 h, and the number of tubes was recorded [
17].
Cell counting kit (CCK)-8 test
EPCs pre-cultured in 96-well plates at 5000 cells/well were added with CCK-8 solution (Dojindo, Kumamoto, Japan) at 0, 24, 48, and 72 h, respectively. Absorbance was measured at 450 nm with a microplate reader (BioTek Instruments, VT, USA).
Flow cytometry
Detection of apoptosis was carried out following the instruction of FITC Annexin V/propidium iodide Apoptosis Detection Kit I (Ribobio, Guangzhou, China). Apoptosis of EPCs was examined on a FACScan flow cytometer (Becton Dickinson, NJ, USA), and data analysis was done by FlowJo software. The upper left quadrant (Q1) was necrotic cells: negative for FITC Annexin V staining and positive for PI staining; the upper right quadrant (Q2) was late apoptotic cells: positive for FITC Annexin V staining and positive for PI staining; the lower right quadrant (Q3) was early apoptotic cells: positive for FITC Annexin V staining and negative for PI staining; the lower left quadrant (Q4) was live cells: negative for FITC Annexin V staining and negative for PI staining [
18].
Transwell detection
EPCs (1 × 10
5) were placed in the top compartment pretreated with Matrigel whereas EBM-2MV supplemented with 20% fetal bovine serum was in the lower compartment. The membrane was stained with 0.1% crystal violet after 24-h cell activity, and the number of EPCs permeating the membrane was calculated under an optical microscope (Olympus) [
19].
Establishment of DVT animal models
Thirty minutes before DVT operation, various constructs at 10 nmol (oe-NC, oe-TUG1, NC antagomir, miR-92a-3p antagomir, NC siRNA, Hmgcr siRNA, oe-TUG1 + NC agomir, oe-TUG1 + miR-92a-3p agomir, miR-92a-3p antagomir + NC siRNA, and miR-92a-3p antagomir + Hmgcr siRNA) were dissolved in 200 μL normal saline and injected into mice through the tail vein. A DVT mouse model was established according to the method previously reported [
20]. In the sham group, occlusion of the femoral veins on both sides was not performed. The mice were euthanized 24 h after the operation, and a fresh thrombus was taken and weighed [
21,
22].
Observation of thrombus
Fresh thrombus was sliced, observed under an optical microscope (XP-330, Shanghai Bingyu Optical Instrument Co., Ltd., Shanghai, China), and pathologically evaluated. 0 point meant no thrombosis, 1 point meant vascular occlusion < 50%, 2 points meant vascular occlusion > 50% (incomplete occlusion), and 3 points meant complete vascular occlusion [
23].
Hematoxylin-eosin (HE) staining
Femoral vein samples of mice were taken, and a 1 cm blood vessel was cut from each vein and fixed with 10% neutral formaldehyde. The blood vessels were paraffin-embedded and cut into 4 μm sections for hematoxylin and 1% eosin staining. After that, images were obtained under an optical microscope (OLYMPUS, Tokyo, Japan) and data analysis was performed by Image-Pro Plus 6.0 software (IPP6.0, Media Cybernetics, MD, USA) [
24].
Quantitative PCR analysis
Trizol (Thermo Fisher Scientific) was adopted to extract the total RNA from cells and tissues. Before reverse transcription, RNase-free DNase I was used to remove possible DNA contamination such as genomic DNA in the extracted total RNA. PrimeScript RT reagent kit (Takara) or microRNA reverse transcription system (GenePharma, Shanghai, China) was implemented to synthesize cDNA. SYBR Green quantitative PCR Master Mix (Takara) or miRNAs Quantitation Kit (GenePharma) was used for RT-qPCR analysis. miR-92a-3p expression was standardized by U6, while other genes were standardized by GAPDH. The 2
^-ΔΔCT method was used to calculate the relative expression of each gene. RT-qPCR primers were supplemented in Supplementary Table
1 [
4].
Immunoblotting analysis
Total protein in tissue and cells was extracted with radio-immunoprecipitation assay lysis buffer, followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Then, a 5% skimmed milk-blocked polyvinylidene fluoride protein membrane (Micropore, Sigma) was incubated with primary antibody against GAPDH (1:1000, Abcam) and Hmgcr (1:1000, Abcam), and with the appropriate secondary antibody. After shooting with the image analysis system (Bio-Rad), the immune complex was visualized using an enhanced chemiluminescence kit (Amsham, UK) [
25].
RNA immunoprecipitation (RIP) assay
EPC lysate collected by RIP lysis buffer (protease inhibitor and RNase inhibitor) was cultivated with protein-G/A plus agarose and Ago2 antibody (Abcam) or immunoglobulin G (IgG; Abcam). The precipitated RNA was extracted using Trizol (Thermo Fisher Scientific) following the manufacturer’s protocol and was subjected to RT-qPCR analysis [
26].
Luciferase reporter assay
Amplified wild type (Wt)-TUG1/mutant (Mut)-TUG1 or Wt-Hmgcr 3’UTR/Mut-Hmgcr 3’UTR sequence was cloned into PGL3 basic vector (Promega, WI, USA). The reporter was co-transfected with NC mimic and miR-92a-3p mimic into EPCs. Luciferase activity was measured using a dual luciferase reporter gene assay system (Promega) after 48 h [
27].
Chromatin immunoprecipitation (ChIP) assay
ChIP was performed via Pierce Sepharose Chip Kit (Thermo Fisher Scientific). Cells were cross-linked in 1% formaldehyde and quenched by glycine solution. The cell pellets were lysed with micrococcal nuclease, and the supernatant was reacted with anti-Hmgcr (1:200, Abcam) or IgG, and precipitated with Protein A/G Sepharose beads (Thermo Fisher Scientific). The protein/DNA complex was eluted by proteinase K, and genomic DNA fragments were analyzed by quantitative PCR.
Statistics
Data were analyzed using SPSS 21.0 (IBM, NY, USA), and the measurement data were represented by mean ± standard deviation. The normal distribution and variance homogeneity were initially measured. As for data fitting normal distribution and variance homogeneity, an unpaired two-tailed Student’s t-test was utilized when comparing two experimental groups, while three experimental groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. P < 0.05 was meaningful for statistical significance.
Discussion
VTE is the third most frequent cardiovascular disease besides myocardial infarction and stroke [
28]. In a cell model and a mouse model, we confirmed that TUG1 overexpression promoted angiogenesis of EPCs and resolution of DVT in mice via miR-92a-3p down-regulation and subsequent Hmgcr up-regulation.
TUG1 is involved in a variety of cell signaling pathways and tissue-specific expressions. Its size is 7.1 kb, which is large enough to fold into a complex secondary/tertiary structure [
29]. It has been indicated that TUG1 expression is constrained in acute lung injury based on a mouse model, and TUG1 overexpression could protect primary murine pulmonary microvascular endothelial cells from lipopolysaccharide (LPS)-induced apoptosis [
30]. For EPCs, TUG1 restoration rescues high glucose-induced decline of cellular migration, invasion, and tube formation abilities, while for diabetic mice, TUG1 overexpression induces angiogenesis in the ischemic limbs [
6]. In a cell model of sepsis, TUG1 up-regulation could present human umbilical vein endothelial cells from LPS-induced apoptosis [
31]. As indicated previously, TUG1 induction is multifunctional regarding proliferation, invasion, and angiogenesis promotion of trophoblasts in the setting of preeclampsia [
32]. Long J et al. have supported that podocyte-specific elevation of TUG1 could improve diabetic nephropathy-associated biochemical and histological features in mice [
33]. In our work, examination of TUG1 expression indicated that TUG1 was poorly expressed in DVT mice. Concerning the action of TUG1, our experimental observations presented that restoration of TUG1 accelerated proliferation, migration, and tube-forming abilities whilst decelerated apoptosis of EPCs; in DVT mice, overexpressed TUG1 exerted to decrease thrombus, and relieve femoral vein pathological damage.
Many articles have demonstrated that TUG1 is a sponge for many miRNAs [
34‐
37]. In the preliminary research, we found that miR-92a-3p was targeted by TUG1 by the bioinformatics website Starbase (
https://starbase.sysu.edu.cn/agoClipRNA.php). Therefore, we chose miR-92a-3p as the research direction. miR-92a-3p is a quantified miRs involved in the regulation of vascular performance, and it is up-regulated in patients with coronary artery disease [
38], as well as in high-density lipoprotein fraction of early diabetic mice after ischemia [
39]. It has been elaborated that miR-92a-3p is associated with endothelial dysfunction, and aberrant elevation of miR-92a-3p expression is recorded in the venous tissue of rats with CRT [
12]. On the other hand, endothelial miR-92a-3p expression is induced after renal injury, and dual suppression of miR-92a-3p/miR-489-3p can relieve atherosclerosis based on a mouse model [
40]. It is well-established that up-regulated miR-92a is involved in endothelial injury, and suppression of miR-92a is feasible to protect endothelial cells in response to acute myocardial infarction [
41]. It is known that hypoxia or high glucose induces injury of EPCs, in which miR-92a suppression could enhance cellular migration and tube formation abilities [
42]. To improve neovascularization, Shyu KG et al. have verified that hyperbaric oxygen has valuable therapeutic effects partly through enhancing lncRNA metastasis-associated lung adenocarcinoma transcript 1-mediated down-regulation of miR-92a [
43]. Based on the sponge adsorption phenomenon between TUG1 and miR-92a-3p, the role of miR-92a-3p was surveyed in detail. The findings displayed that miR-92a-3p inhibition similarly phenocopied the impacts of overexpressed TUG1 on EPCs in vitro and DVT mice in vivo.
Angiogenesis is regulated by the Hmgcr pathway through the differentially regulated arteriovenous demand for protein prenylation in endothelial cells [
14]. Hmgcr function impairment disturbs the stability of cerebral blood vessels, leading to the progressive expansion of blood vessels and subsequent rupture of blood vessels [
44]. From our analysis, it was noticeable that miR-92a-3p had a targeted regulatory effect on Hmgcr expression. Given that, we subsequently found that Hmgcr suppression blocked EPCs’ activities, and aggravated DVT in mice.
All in all, our study analysis exhibited the TUG1-induced protection against DVT through interacting with miR-92a-3p and Hmgcr. This research has delved into a brand-new way to treat DVT from the molecular TUG1/miR-92a-3p/Hmgcr cascade. One of the study limitations is that the signaling pathway involved in the TUG1/miR-92a-3p/Hmgcr axis-regulated DVT resolution was not explored. Additionally, TUG1 is a sponge of the many miRNAs, and miR-92a-3p is not the only miRNA responsible for the phenotype observed in this study, therefore, further research is warranted to validate our findings.
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