BM-derived EPCs isolation and expansion
Sprague-Dawley (SD) rats (male, 150g, SCXK2016-0006) from Charles River Laboratories Supplier in China (Beijing, China, SCXK2016-0006) were sacrificed by cervical dislocation. The rats’ tibias and femurs of both sides (4 long bones from the hind limbs) were rinsed repeatedly to obtain the bone marrow. To isolate the mononuclear cells (MNCs), Histopaque-1083 (Sigma-Aldrich, St. Louis, MO, USA) was put into use when dealing with the cell suspension with density gradient centrifugation. After isolating, mononuclear cells were seeded on a 100-mm plate at a density of 2.5×106 cells/cm2. These plates were coated at 37℃ using fibronectin (R&D Systems, Minneapolis, MN, USA) 24 hours before. Cells were cultured with EGM-2 (Lonza, Walkersville, MD, USA) containing 5% fetal bovine serum. 24 hours later, gently suck the cells floating in the medium and discard them. Changed the medium daily for the first 3 days and then every 2 days. About 5 days later, cell clusters could be observed. Cells were examined under the microscope every day (IX73, Olympus, Center Valley, PA, USA) (Fig. 1A-D). A colony forming unit consists of a circular central core surrounded by elongated and spindle cells could be identified as endothelial progenitor cell colony[11]. EPCs at passage 3 were obtained and used in the further experiment.
Incorporation of Dil-Ac-LDL and lectin binding
After 8 days of culture, washed the EPCs extensively with phosphate buffer solution and subsequently stained with 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (Dil-ac-LDL, Maokang Biotechnogy Co., Ltd., Shanghai, China, MP6013) at a final concentration of 10mg/L. Before Fixing the cells with 2% paraformaldehyde in PBS for 10min, the cells were incubated at 37℃ and 5% CO2 for 4h. After fixation, dyed the cells with fluorescein isothiocyanate-labeled Ulex europaeus agglutinin (FITC-UEA-1) (FITC-UEA-1, Maokang Biotechnogy Co., Ltd., Shanghai, China, MP6308) at a final concentration of 10mg/L. The cells were observed under the confocal fluorescent microscopy (TCS SP5 II), cells double positive for Dil-Ac-LDL and FITC-UEA-1 staining were identified as EPCs (Fig. 1E-G).
Animals and protocols
All animals were kept free of pathogen and provided clean water and rats diet for maintaining ad libitum. The final sample size was determined based on the preliminary results. SD rats (weight: 250-300g, age: 7-8 weeks, male) were obtained from the Charles River Laboratories Supplier in China (Beijing, China, SCXK2016-0006). The rats were anaesthetized with 40mg/kg bw pentobarbital via intraperitoneal injection and subject to balloon injury in the common carotid artery of the right side. Briefly, the rats were placed in supine position, the bifurcation of the right carotid artery was exposed after intravenous injection of 100 U/kg of heparin sodium via a neck midline incision. Prepared two ligatures around the external carotid artery and one ligature around the internal carotid artery. First, tied off the distal ligature around the external carotid artery to ensure that the blood flow is blocked. After that, make a temporary occlusion to the internal carotid artery and proximal side of common carotid artery, performed a transverse arteriotomy between the proximal and distal ligatures of the external carotid artery. Inserted a balloon angioplasty catheter from the incision in artery. The balloon was then inflated at 2-3 atm and passed through the artery for 3 times to ensure uniformity of the extent of the endothelium injury. After injury, tied off the proximal ligature of the external carotid artery as soon as the catheter was removed. Then the blood flow through the common and internal carotid arteries was restored. Sutured the skin with 4/0 silk. Rats received either 1×106 bone marrow-derived EPCs labeled by PKH-26 via intravenous tail vein injection after injury (Fig. 1H-J). Control group received a corresponding amount of EGM-2 medium. Animals were allowed to recover, Ibuprofen (15mg/kg/day) dissolved in water was provided ad libitum as a pain killer for 3 days after the balloon-injured procedure. At 1 day, 4 days, 7 days, and 14 days after balloon injury, rats were sacrificed and the common carotid arteries were harvested.
Perfusion and tissue preparation
The rats were anaesthetized with 40mg/kg bw pentobarbital via intraperitoneal injection and received 10mg/kg bw 0.1% Evans blue dye (Coolaber) by intravenous tail vein injection 30 minutes before tissue harvesting. Perfused the animals transcardially follow the sequence of 0.01M phosphate-buffered saline (PBS) (P3813, Sigma-Aldrich) and 4% paraformaldehyde (PFA; 158127, Sigma-Aldrich) in PBS. The common carotid arteries were harvested and fixed in 4% PFA at 4℃ at least 24 hours before clearing.
Tissue clearing procedure
The common carotid artery embedded in 1% agar was dehydrated with methanol solutions (mixed with PBS) follow the sequence of concentrations 25, 50, 75, and 100 volume % for 3 hours each. Benzyl alcohol and benzyl benzoate was used in a ratio of one to three as a refractive index matching solution. All steps were performed at room temperature and keep shaking slightly. Finally, placed the sample in the dark by covering aluminum foil. The artery was observed by light-sheet microscopy.
Light-sheet microscopy
Arteries were observed under the light-sheet fluorescence microscope (LaVision Biotec, Bielefeld, Germany). We used a 2.5x objective lens (Mv PLAPO 2VC, Olympus) or a 4x objective lens (Mv PLAPO 2VC, Olympus) covered with a 6mm working distance dipping cap. A supercontinuum white light laser (SuperK EXTREME 80 mHz VIS with wavelength from 400 to 2400nm, NKT Photonics, Cologne, Germany) was chosen as a laser source. To observe the cell distribution and artery morphology, the filters were set as 551/40nm excitation and 567/50nm emission for PKH26 and 640/30nm excitation and 690/50nm emission for Evans blue. The step size was set to 5µm. Carotid artery scanning range was set to 1mm. 3D projections of the tagged image file format (TIFF) images of the artery were obtained by using Imaris software (Bitplane, Oxford Instruments Company).
Image Processing
Images of light-sheet fluorescence microscopy were stored as 16 bit ome.tif stacks. Considering that the range of pixel values of different images is inconsistent, we firstly normalize all the image values to [0,255]. Secondly, the threshold value is obtained by using the non-invasive group, which is the maximum value of the image. Then, images of other groups are processed, with the pixel value less than the threshold setting to zero. The average value without zero is the sum of processed pixels divided by the number of non-zero pixels. All the image values would be normalized to [0,1] before the final statistical analysis. Images of hematoxylin and eosin (H&E) and immunofluorescence were analyzed using tools of ImageJ (version 1.52a, Wayne Rasband, National Institutes of Health, USA).
Morphometric analysis
Carotid arteries were trimmed and paraffin embedded in the Histology and Comparative Pathology Facility. Paraffin blocks were trimmed with a microtome until a full cross-section of the artery was visualized. Tissue sections of 5μm thick from three different regions of the artery were collected. Arterial sections were dyed with H&E and photographed with a Leica DM3000 microscope. For immunofluorescence, artery sections were deparaffinized, rehydrated, and antigen retrieved by a sodium citrate method (Vector Labs H-3300). Arteries were blocked with PBS containing 0.3% triton-X-100 and 5% normal goat serum for one hour at room temperature, and incubated with primary antibodies in PBS containing 0.3% triton-X-100 and 2% bovine serum albumin overnight at 4℃. After washing, sections were incubated with fluorochrome-conjugated secondary antibodies for one hour at room temperature. After washing, arterial sections were stained with DAPI (4',6-diamidino-2-phenylindole) using FLUORO-GEL II with DAPI (Beyotime, C1005). Images were taken with a Leica DM3000 microscope. Antibodies used were Rabbit anti-CD31 antibody(Abcam, ab24590, 1:50 dilution), and goat anti-rabbit IgG (Boster, BA1032, 1:400 dilution).
Statistical analysis
Continuous variables consistent a normal distribution were expressed as mean ± standard deviation (SD). Multiple group comparisons were performed using a one-way ANOVA, followed by a post hoc analysis using the least significant difference (LSD) t-test or Dunnett’s T3 post-hoc test using Welch’s ANOVA. Comparisons between two independent groups were analyzed using Student’s t-test. Two-sided tests were used throughout the experiment. P < 0.05 was considered statistically significant. ALL data were analyzed using GraphPad Prism-8 statistic software (La Jolla, CA).