MicroRNA design
In order to evaluate the angiogenic potential of miR-126, shRNA-lentiviral vector construct was made. At first step shRNA was designed. In the present study, annealed oligos method was used. However, some changes were made in the shRNA design according to the purpose of the study. The entire procedure involves the following steps:
1. The sequence of miR-126-3p and miR-126-5p was determined according to sequences from http://www.mirbase.org/. The 3p arm of shRNA is known as guide strand and the 5p arm is known as passenger strand. The shRNA sequence (from 5′ to 3′) will be in the order of passenger strand, loop, and then guide strand.
MiR-126-3p sequence: 5´-UCGUACCGUGAGUAAUAAUGCG-3´
MiR-126-5p sequence: 5´-CAUUAUUACUUUUGGUACGCG-3´
2. A sixth nucleotide loop was set to connect 3p and 5p strands.
Loop sequence: CTCGAG
3. In order to clone the shRNA to pLKO.1-TRC vector the complementary sequences of restriction endonuclease Age1 and EcoR1 enzymes were designed at the 5´ ends of the oligonucleotides.
4. According to the mentioned points the sequence of the two oligos will be as follows:
Forward oligo: 5’ CCGG—21bp 5p strand—CTCGAG—21bp 3p strand—TTTTTG 3’
Reverse oligo: 5’ AATTCAAAAA—21bp 5p strand—CTCGAG—21bp 3p strand 3’
BM-MSCs isolation and culture
To isolate bone marrow, 6-week-old C57BL/6 mice were killed by cervical dislocation, then the ends of tibia and femur bones were cut to expose the marrow. 5-ml syringe containing complete media used to extract the cells via flushing the marrow plug out of the cut end of the bone with 1 ml of complete media and collect in a 15-ml tube. Strong flushing is necessary during marrow cell preparation. Then the cell pellet derived from 2 tibia and 2 femur bones was suspended in growth medium containing Dulbecco’s modified Eagle’s medium (DMEM) high glucose (Gibco, USA) and 10% fetal bovine serum (FBS; Gibco, USA) with 100 U/mL penicillin–streptomycin (Gibco, USA) and cultured in a 75-cm2 culture flask and were maintained at 37C and 5% CO2. Nonadherent cells were removed after 24 h and the flask was washed with phosphate-buffered saline (PBS; Gibco, USA). The medium was changed regularly every 3-4 days, and at approximately 70% confluence, the cells were detached using trypsin-EDTA (Gibco, USA) and transferred to new flasks, these cells were considered as passage 1(Fig. 1).
Differentiation of BM-MSCs
Osteogenesis: passage 3 BM-MSCs were incubated to differentiate into osteoblasts in corresponding induction medium for 2 weeks. The osteogenic medium contained DMEM, 15% FBS (Gibco, USA), 100 µM L-ascorbic acid, 10 mM glycerol 3-phosphate, and 100 nM dexamethasone (Sigma-Aldrich). After 2 weeks the cultured cells were stained with Alizarin Red solution to visualize calcium deposits. The cells were fixed in 4% paraformaldehyde (PFA) for 20 minutes and stained using Alizarin Red solution for 20 minutes at room temperature.
Adipogenesis: passage 3 BM-MSCs were incubated to differentiate into adipocytes in corresponding induction medium for 2 weeks. The Adipogenic medium contained DMEM, 15% FBS, 100 µM L-ascorbic acid, 200 μM Indomethacin, 1000nM insulin (Sigma-Aldrich) and 100 nM dexamethasone (Sigma-Aldrich). After 2 weeks the cultured cells were stained with Oil Red-O solution to visualize lipid vacuoles. The cells were fixed in 4% paraformaldehyde (PFA) for 20 minutes and stained using Oil Red-O solution for 20 minutes at room temperature.
Flow cytometry analysis of MSCs
For the analysis of surface marker expression of BM-MSCs Flow cytometry was performed. The expression of surface markers was evaluated using monoclonal antibodies against mouse anti-CD44 FITC, anti-CD105 perCP, anti-CD45 FITC, and anti-CD34 PE. The cells at passage 4 were used. The data was analyzed with Flow Jo software (version 7.6).
Lentivirus production and titration
In the present study we used second generation self-inactivating (SIN) human immunodeficiency virus-1-based (HIV-1), VSV-G pseudotyped lentiviral vectors. Second generation LVs were produced by calcium phosphate-mediated transfection into 293T cells. Briefly, packaging plasmid psPAX2 (Addgene plasmid 12260), envelope plasmid pMD2.G (Addgene plasmid 12259) and transfer plasmid pLKO.1-TRC (Addgene plasmid 10878) were transfected into 293T cells using Ca3PO4 transfection protocol. Culture medium was replaced 6 h post-transfection. Vector-containing supernatants were collected 24, 48 h, and 72 h post-transfection, and concentrated by Amicon filter 100 MW. Lentivirus vector titer was determined on 293T cells and vector particles were measured by puromycin titration. Vector aliquots were stored at -80°C.
Transduction of BM-MSCs and puromycin reporter gene detection
Cells were seeded in six-well plates at 5 × 104 cells per well and different concentrations of virus were added and incubated at 37°C for 72 h. The volume of lentivirus to be used was determined from titer results. After incubation, 1.5 mL of fresh DMEM complete containing 2µg puromycin (Sigma) was added to each well. It was the beginning of the selection process. After 2 days, the culture medium was removed from each well and fresh medium was added without puromycin. The resistant cells (stable cell line) were cultured and expanded into T75 flask.
Cell migration assay
Wound-healing assay on endothelial cells, human umbilical vein endothelial cell (HUVECs), was used to study the paracrine effect of BM-MSCs and BM-MSCs miR-126 on endothelial cell migration potential. After 24-h FBS starvation of MSCs, the supernatants from cells were collected. The monolayer HUVEC cells were scratched using a sterile plastic micropipette tip. The cells were washed by DMEM and PBS twice. After that, wound healing initiated by adding the collected supernatants from BM-MSCs and BM-MSCs miR-126 on HUVEC cultures in different wells of a 6-well plate. After incubating the cells at 37 °C in 5% CO2 for 0, 15, 18, 20, 22 and 24 h, the migration images of the cells were observed under a microscope with 10x (Nikon, ECLIPSE, TS100), the distance of cell migration was measured and images taken using image-J Analysis software.
Mouse Critical limb Ischemia model
All animal experiments were performed according to the guidelines approved by the Shiraz University of Medical Sciences ethics committee (1397.430).
Male C57BL/6 mice, weighing 25–30 g (6–8 week-old) purchased from Comparative and Experimental Medicine center at Shiraz University of Medical Sciences and were selected randomly. Animals were kept in standard conditions (12 h light and 12 h darkness, temperature of 18–22 °C, and 55 ± 5% humidity). They were fed standard mouse diet and given water ad libitum.
For creating critical limb ischemia (CLI) model, all animals in each group were anesthetized intraperitoneal (IP) administration using ketamine 10% (100 mg/kg, Alfasan CO., Netherlands) and xylazine 2% (5 mg/kg, Alfasan CO., Netherlands). Animals were placed in dorsal recumbency. A skin incision (about 10 mm) was done along the center of the medial thigh from the abdomen towards the knee. Subcutaneous fat tissue superficial was gently pushed away to exposed femoral neurovascular bundle. The femoral artery pushed away from the femoral vein and nerve. Subsequently, the femoral artery was isolated by blunt dissection from the femoral vein at the ligation sites between the proximal caudal femoral artery and the bifurcation of the deep femoral artery and saphenous artery. Then two sutures were passed using 6-0 silk, transected and cauterized. The incision was closed using continues 5-0 vicryl sutures. The operation was done under a surgical microscope (Zeiss OP-MI6 SD; Carl Zeiss, Goettingen, Germany) (Fig. 2).
Animal groups and transplantation
The male mice were randomly divided into the following four groups (n = 12/group): (1) control group: CLI was performed and received PBS into ischemic site, (2) BM-MSCs group: CLI was performed and received BM-MSCs, (3) virus-miR-126 (recombinant vector) group: CLI was performed and received recombinant vector, (4) BM-MSCs miR-126 group: CLI was performed and received BM-MSCs that transfected with virus-miR-126. To transfer the cells and recombinant vector into ischemic leg, intramuscularly (IM) injection of 5×105 BM-MSCs and BM-MSCs miR-126 that resuspended in PBS and 4.7×10 6 virus particles as treatment groups and PBS as a control group were done at four different sites of gastrocnemicus (GC) muscle 24 hours after surgery.
Additionally to identify the presence of grafted BM-MSCs and BM-MSCs miR-126 in the gastrocnemicus (GC) muscle the fifth and sixth groups were added as, survival groups: CLI was performed on female mice (n = 5/group) and received intramuscularly (IM) injection of 5×105 male BM-MSCs and BM-MSCs miR-126 at four different sites of gastrocnemicus (GC) muscle 24 hours after surgery.
Functional scoring
Functional grading was performed according to the Tarlov, ischemia, modified ischemia, function and the grade of limb necrosis scoring system at pre-operation, post-operation and at days 3, 7, 14, 21 and 28 after transplantation. The degree of ischemic damage was evaluated through indicators, such as skin color changes, swelling, and grade of the limb necrosis (Table 1).
Table 1
Tarlov score
|
Description
|
0
|
No movement
|
1
|
Barely perceptible movement, non–weight bearing
|
2
|
Frequent movement, non–weight bearing
|
3
|
Supports weight, partial weight bearing
|
4
|
Walks with mild deficit
|
5
|
Normal but slow walking
|
6
|
Full and fast walking
|
Ischemia score
|
Description
|
0
|
Autoamputation > half lower limb
|
1
|
Gangrenous tissue > half foot
|
2
|
Gangrenous tissue < half foot, with lower limb muscle necrosis
|
3
|
Gangrenous tissue < half foot, without lower limb muscle necrosis
|
4
|
Pale foot or gait abnormalities
|
5
|
Normal
|
Modified ischemia score
|
Description
|
0
|
Autoamputation of leg
|
1
|
Leg necrosis
|
2
|
Foot necrosis
|
3
|
Discoloration of 2 toes
|
4
|
Discoloration of 1 toe
|
5
|
Discoloration of > 2 nails
|
6
|
Discoloration of 1 nail
|
7
|
No necrosis
|
The grade of limb necrosis
|
Description
|
0
|
Normal limb without swelling, necrosis or atrophy of muscle
|
1
|
Necrosis limiting to toes (toes loss)
|
2
|
Necrosis extending to a dorsum pedis (foot loss)
|
3
|
Necrosis extending to a crus (knee loss)
|
4
|
Necrosis extending to a thigh (total hind-limb loss)
|
Function score
|
Description
|
0
|
Dragging
|
1
|
No plantar flexion
|
2
|
No toe flexion
|
3
|
No grabbing force
|
4
|
Some grabbing force
|
5
|
Normal
|
Investigation of donor cell survival
To identify the presence of grafted male cells in the female gastrocnemicus (GC) muscle (the survival groups), the Y chromosome–specific SRY gene was assessed by polymerase chain reaction. At days 3, 7, 14, 21, and 28 after treatment, the GC muscle was collected, genomic DNA was extracted using Trizol (Sigma-Aldrich) and then SRY analysis was performed by PCR. The forward primer sequence was 5′-TTTATGGTGTGGTCCCGTGGTGAG-3′ and the reverse primer sequence was 5′-TTGGAGTACAGGTGTGCAGCTCTAC-3′.
RNA isolation and quantitative real-time PCR
In order to evaluate the expression of target genes (VEGF, SPRED1, PIK3R2 and GAPDH as housekeeping gene), after transplantation at days 7, 14, 21 and 28, the GC muscle was collected, total RNA was extracted using Trizol (Sigma-Aldrich T9424) according to the manufacturer’s protocol. The concentration of total RNA was quantified by the absorbance at 260 nm. Extracted RNA was then reverse-transcribed using the SMOBIO cDNA Synthesis Kit RP1300. Then gene expression analysis was performed by Real-time PCR. qRT-PCR was performed on an ABI Prism 7500 PCR system using RealQ Plus 2X Master Mix Green Low Rox (Amplicon). PCR was performed in a total reaction volume of 15 ml consisting of appropriate cDNA. For semi-quantitative RT-PCR, the following primers were used (Table 2).
Table 2
Primers used for quantitative real-time PCR
GAPDH forward
|
5′-CTGTGGGCAAGGTCATCCCAGAG-3′
|
GAPDH reverse
|
5′-CTTCTTGATGTCATCATACTTGGCAGGTT-3′
|
VEGF forward
|
5′-TACCTCCACCATGCCAAGTG-3′
|
VEGF reverse
|
5′-AAGATGTCCACCAGGGTCTC-3′
|
PIK3R2 forward
|
5′-GCTTCTCAGAGCCCCTTACCTTCTG-3′
|
PIK3R2 reverse
|
5′-GTCCTCCTTCACCACCTGGTCTTGT-3′
|
SPRED1 forward
|
5′-ACTTCCCGTTCCCTAGTGAAAGATCAC-3′
|
SPRED1 reverse
|
5′-AGCCTTGCTGACTGAATGGTATCTGG-3′
|
Tarlov score
|
Description
|
0
|
No movement
|
1
|
Barely perceptible movement, non–weight bearing
|
2
|
Frequent movement, non–weight bearing
|
3
|
Supports weight, partial weight bearing
|
4
|
Walks with mild deficit
|
5
|
Normal but slow walking
|
6
|
Full and fast walking
|
Ischemia score
|
Description
|
0
|
Autoamputation > half lower limb
|
1
|
Gangrenous tissue > half foot
|
2
|
Gangrenous tissue < half foot, with lower limb muscle necrosis
|
3
|
Gangrenous tissue < half foot, without lower limb muscle necrosis
|
4
|
Pale foot or gait abnormalities
|
5
|
Normal
|
Modified ischemia score
|
Description
|
0
|
Autoamputation of leg
|
1
|
Leg necrosis
|
2
|
Foot necrosis
|
3
|
Discoloration of 2 toes
|
4
|
Discoloration of 1 toe
|
5
|
Discoloration of > 2 nails
|
6
|
Discoloration of 1 nail
|
7
|
No necrosis
|
The grade of limb necrosis
|
Description
|
0
|
Normal limb without swelling, necrosis or atrophy of muscle
|
1
|
Necrosis limiting to toes (toes loss)
|
2
|
Necrosis extending to a dorsum pedis (foot loss)
|
3
|
Necrosis extending to a crus (knee loss)
|
4
|
Necrosis extending to a thigh (total hind-limb loss)
|
Function score
|
Description
|
0
|
Dragging
|
1
|
No plantar flexion
|
2
|
No toe flexion
|
3
|
No grabbing force
|
4
|
Some grabbing force
|
5
|
Normal
|
GAPDH forward
|
5′-CTGTGGGCAAGGTCATCCCAGAG-3′
|
GAPDH reverse
|
5′-CTTCTTGATGTCATCATACTTGGCAGGTT-3′
|
VEGF forward
|
5′-TACCTCCACCATGCCAAGTG-3′
|
VEGF reverse
|
5′-AAGATGTCCACCAGGGTCTC-3′
|
PIK3R2 forward
|
5′-GCTTCTCAGAGCCCCTTACCTTCTG-3′
|
PIK3R2 reverse
|
5′-GTCCTCCTTCACCACCTGGTCTTGT-3′
|
SPRED1 forward
|
5′-ACTTCCCGTTCCCTAGTGAAAGATCAC-3′
|
SPRED1 reverse
|
5′-AGCCTTGCTGACTGAATGGTATCTGG-3′
|
The two-step reaction was performed triplet for all samples. The PCR was performed under the following cycling conditions: denaturation at 95°C for 15 min, followed by 40 cycles of 30 sec at 95°C and 30 sec at 62°C (annealing), and extension at 72°C for 30 sec. Data were analyzed by the comparative cycle threshold (Ct) method and normalized against GAPDH controls.
The two-step reaction was performed triplet for all samples. The PCR was performed under the following cycling conditions: denaturation at 95 °C for 15 min, followed by 40 cycles of 30 sec at 95°C and 30 sec at 62°C (annealing), and extension at 72 °C for 30 sec. Data were analyzed by the comparative cycle threshold (Ct) method and normalized against GAPDH controls.
Histopathological evaluation
Histological analysis of limb tissues were done at days 7, 14, 21 and 28 after transplantation. To evaluate that the target cells (BM-MSCs and BM-MSCs miR-126) and recombinant vector can induce angiogenesis in the gastrocnemius muscle, hematoxylin-eosin (H&E) and immunohistologic staining (with an anti-CD31 antibody) were performed (in the previous study were described). Then the tissue slides were evaluated for muscle regeneration and microvessel density (by MVD score).
Statistical analysis
Statistical analysis was performed using GraphPad Prism 7 Software. Results are expressed as the mean standard error of the mean. Comparisons between multiple groups were performed using one-way analysis of variance (ANOVA), with post hoc testing performed with Bonferroni analysis or unpaired t-tests, as appropriate. Also comparisons between two groups were performed using independent sample T-test. P values ≤ 0.05 were considered statistically significant (*p < .05, **p < .01, ***p < .001, ****p < .0001). Data were presented as mean ± standard deviation (SD).