Animal models construction
Male Sprague–Dawley (SD) rats (200–250 g) were purchased from Shanghai Laboratory Animal Center (Shanghai, China) and were kept in specific pathogen-free (SPF) animal rooms. All the procedure was approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Soochow University. This rat model is well described in previous studies . In brief, the rats were anesthetized by intraperitoneal injection of 7% pentobarbital and underwent midline laparotomy in order to dissect the inferior vena cava (IVC) free from aorta. And then IVC was ligated just below the upper renal vein with a 6-0 Prolene sutures. The posterior venous branches were also tightened. After that, the confluence of iliac vein was clamped with vascular clips for 15 min. Then the incision was closed and the rats were allowed to recover after surgery. Thirty rats were randomly divided into three groups (n = 10 for each group): (A) blank control group received 1 ml EGM-2-MV medium, (B) lentivirus vector group received blank vector and (C) miR-21 overexpression group received pGLV3-H1-Puro-miR-21.
Cell isolation and culture
EPCs isolation was performed as previously described . Briefly, male SD rats (200–250 g) were sacrificed and bone marrow was harvest from femurs and tibias. Mononuclear cells were acquired with density gradient centrifugation and cultured in EGM-2-MV (Lonza, MD, USA) medium at 37 °C in a 5% CO2 incubator. Non-adherent cells were washed 4 days after culture. Then medium was changed every 2 days. EPCs in passage 3–4 were used for subsequent experiments.
Seven milliliters of venous blood were collected from DVT patient from July 2012 to June 2015 in our department. Inclusion criteria consisted mainly age range 30–50 years, suffered from primary acute DVT extending to the high femoral or iliac vein, symptom duration of less than 2 weeks, verified by ultrasound or digital subtraction angiography (DSA), good functional status. The following exclusion criteria were applied: isolated infrapopliteal thrombosis, contraindications to anticoagulation or thrombolytic agents, with malignant tumors, bacterial endocarditis, during pregnancy and declined to provide informed consent. The whole blood was centrifuged at 4 °C, 2000 rpm for 5 min followed by centrifugation at 12,000 rpm for 15 min. The serum samples were portioned in aliquots and stored at − 80 °C. The protocols were approved by the Institutional Review Board of Second Affiliated Hospital of Soochow University. Informed consent was obtained from each participant prior to specimen acquisition.
Serum RNA isolation
Small RNAs were extracted from 500 μL of serum using a miR-PARIS kit (AM1556) according to the manufacturer’s instructions. To allow for normalization of sample-to-sample variation in RNA isolation, synthetic Caenorhabditis elegans miRNAcel-miR-21 (purchased as a custom RNA oligo nucleotide from Qiagen) was added (50 pmol/L in a 5 μL total volume) to each denatured sample.
Quantitative real-time PCR (RT-PCR) analysis
Total RNA of EPCs was extracted with Trizol Reagent (Invitrogen; Carlsbad, CA, USA). Mature miRNA expression analysis was done using the miRNA real-time PCR quantitation kit (Applied Biosystems, Foster City, CA, USA). The expression of miR-21 was carried out using the Applied Biosystems 7500 Real Time PCR System, with U6 as an internal control. mRNA expression analysis was performed using Power SYBR Green (Applied Biosystems, Foster City, CA, USA). PCR primers (forward and reverse) were as follows: rno-mir-21, forward: GCGGCGGTAGCTTATCAGACTG and reverse: ATCCAGTGCAGGGTCCGAGG; U6, forward: GCTTCGGCAGCACATATACTAAAAT and reverse: CGCTTCACGAATTTGCGTGTCAT; GAPDH, forward: CGCATCTTCTTGTGCAGTG and reverse: GAGGGTGCAGCGAACTTTATT.
To regulate the expression of miR-21 in EPCs, miR-21 agomir, antagomir or respective negative control were transfected into cells with Lipofectamine 3000 (Invitrogen; Carlsbad, CA, USA). 72 h after transfection, cells were harvested for subsequent experiments. Transfection efficacy was evaluated by qRT-PCR. The sequence of miR-21 agomir were: 5′-UAGCUUAUCAGACUGAUGUUGA-3′; agomir negative control were: 5′-UUCUCCGAACGUGUCACGUTT-3′; miR-21 antagomir were: 5′-UCAACAUCAGUCUGAUAAGCUA-3′; antagomir negative control were: 5′-CAGUACUUUUGUGUAGUACAA-3′. The target sequence of siRNA against rat FASLG (NM_012908.1) was 5′-GCAGAACUCCGAGAGUCUATT-3′.
A total of 1 × 104 cells of EPCs were seeded to 24-well plates in a final volume of 800 ul medium for assessment of proliferation ability. 72 h after seeding, cell proliferation was evaluated using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). All experiments were performed in triplicate.
In vitro tube formation assay
For in vitro tube formation assay, EPCs were seeded in the presence of EGM-2-MV medium at a density of 2 × 104/mL for 8 h at 37 °C in a 48-well plate coated with Matrigel (R&D Systems, MN, USA). The formation of capillary-like structures was captured under a light microscope. Each experiment was done in triplicate.
The 3′-UTR of FASLG containing the putative miRNA target site(s) was cloned into the SpeI and HindIII sites of the pMIR-REPORT Luciferase vector (Ambion, TX, USA). 293T cells were transfected with firefly luciferase reporter vector, miRNA, and renilla luciferase control vector using lipofectamine 3000. The reporter assays were analyzed by the examination of ratio between firefly and renilla luciferase activities. The experiments were performed in triplicate.
Total proteins extracted from EPCs using RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) were separated by SDS-polyacrylamide gel and transferred into PVDF membranes. Membranes were blocked with 5% non-fat milk TBST and incubated with primary antibody for FASLG (Abcam, Cambridge, MA, USA), followed by the incubation with appropriate HRP-conjugated secondary antibody. β-actin (Sigma-Aldrich, St. Louis, MO, USA) was then measured as internal control. The densitometry of western blot results was measured using ImageJ software.
Generation of recombinant lentivirus miR-21 and injection
The lentiviral expression vector pGLV3-H1-Puro-miR-21 was constructed to stably express miR-21 in EPCs. 293T cells were cotransfected with pGLV3-H1-Puro vector or pGLV3-H1-Puro-miR-21 plasmid using lipofectamine 3000 (Invitrogen; Carlsbad, CA, USA). Then viral particles were harvested from 293T cells and enriched. Finally, viral titers were determined by counting the labeled cells or using qRT-PCR to detect GFP expression. Lentivirus miR-21 or lentivirus vector was used to transfer miRNA into rats. Three days after thrombus formation, rats were injected within the thrombus with different solution. The solution contained 1 × 109 TU/mL lentivirus miR-21 or lentiviral particles. The rats in normal group were injected with 2 mL EGM-2-MV medium.
Seven days after injection, the rats were perfused and IVC segments with thrombus were removed and fixed in 4% paraformaldehyde, embedded in paraffin. All the fixed tissue was sliced at 8-μm intervals. Hematoxylin/eosin staining were done using standard procedures. Images were captured using an inverted microscope. Before the thrombi were weighed, excessive blood on the thrombi was removed by filter paper.
Digital subtraction angiography (DSA)
Seven days after injection, IVC venography was acquired with digital subtract angiography (DSA, GE Innova 3100, USA) by injecting contrast media into rat caudal vein or femoral vein to determine the recanalization and resolution of thrombus in vivo. All the acquired images were analyzed using image J software.
Patients attended follow-up visits at 1 month and 3 months after treatment and every 6 months thereafter and were contacted by telephone or e-mail at the 3-month mark between visits. The primary outcome of the study was either a recurrence of venous thromboembolism or PTS. Recurrent DVT was defined as a composite of symptomatic, objectively confirmed deep-vein thrombosis, nonfatal pulmonary embolism, or fatal pulmonary embolism. PTS was defined as patients with suggested symptoms including pain, heaviness, edema, varicose vein, discoloration and or ulcer in affected lower extremity. Villalta score was recorded to assess the severity of PTS during follow-up.
Data are presented as mean ± SEM. Differences among groups were tested by one-way ANOVA. Statistical analyses between two groups were evaluated based on the Student’s two-tailed t- test. Kaplan–Meier method was used to compare the recurrent DVT between patients in different groups with log-rank test. Univariate associations between candidate predictors and recurrence of DVT were examined with 95% confidence interval (CI) by using Cox proportional hazards model. Multivariate Cox regression analysis with backward conditional method was performed to select significant prognostic factors. In all analyses, p < 0.05 was considered significant.