Netrin‐1 Promotes Inflammation Resolution to Achieve Endothelialization of Small‐Diameter Tissue Engineering Blood Vessels by Improving Endothelial Progenitor Cells Function In Situ

Abstract The transplant of small‐diameter tissue engineering blood vessels (small‐diameter TEBVs) (<6 mm) in vascular replacement therapy often fails because of early onset thrombosis and long‐standing chronic inflammation. The specific inflammation state involved in small‐diameter TEBVs transplants remains unclear, and whether promoting inflammation resolution would be useful for small‐diameter TEBVs therapy need study. The neural protuberant orientation factor 1 (Netrin‐1) is found present in endothelial cells of natural blood vessels and has anti‐inflammatory effects. This work generates netrin‐1‐modified small‐diameter TEBVs by using layer‐by‐layer self‐assembly to resolve the inflammation. The results show that netrin‐1 reprograms macrophages (MΦ) to assume an anti‐inflammatory phenotype and promotes the infiltration and subsequent efflux of MΦ from inflamed sites over time, which improves the local microenvironment and the function of early homing endothelial progenitor cells (EPCs). Small‐diameter TEBVs modified by netrin‐1 achieve endothelialization after 30 d and retain patency at 14 months. These findings suggest that promoting the resolution of inflammation in time is necessary to induce endothelialization of small‐diameter TEBVs and prevent early thrombosis and problems associated with chronic inflammation. Furthermore, this work finds that the MΦ‐derived exosomes can target and regulate EPCs, which may serve as a useful treatment for other inflammatory diseases.

2 cervical dislocation. Peritoneal MΦ were obtained and cultured as previously described. Briefly, we injected rats with sterile phosphate buffer saline (PBS) containing penicillin and streptomycin, gently massaged the belly, then aspirated the fluid, and pelleted and resuspended the cells in red blood cell lysis buffer for 1-2 minutes. After centrifugation, cells were cultured in RPMI medium 1640 containing 10% FCS. Nonadherent cells were removed by washing 2-3 h later, and fresh medium was added.

EPCs isolation and culture
Mononuclear cells were isolated from the peripheral blood of SD rats (n=10) via gradient density centrifugation, seeded onto 24-well plates, and cultured at 37°C and 5% CO 2 in media containing 10% FBS, vascular endothelial growth factor (R&D Systems; 10 ng/ml), basic fibroblast growth factor (R&D Systems; 3 ng/ml), heparin (Abcam; 90 mg/ml), penicillin (Abcam; 100 U/ml), streptomycin (Abcam; 100 U/ml), and fungizone (Abcam; 0.25 mg/ml) for 24 h. [1] Next, the medium was changed to remove non-adherent cells and changed again every other day. Early EPCs were harvested after 7 d of culture, and late EPCs were harvested after 2-3 weeks of culturing. [2] Spleens from SD rats (n=10) were minced into 1-mm³ fragments, ground into solution, and then sequentially passed through 100-and 40-μm mesh. Red blood cells in the resulting renal cells were lysed using red blood cell lysis buffer, and EPCs were isolated and cultured as procedure for isolating EPCs from peripheral blood (PBL) described above. 3

Exosomes purification
The MΦ-exosomes isolation procedure was performed as previously described. [3] Briefly, 2 ml serum-free RPMI medium was used for culturing MΦ in each group. After 48 h, cell culture supernatants were enriched by using a Total Exosome Isolation (from cell culture media) kit (Life Technologies, MA, USA) following the manufacturer's instructions (n=10).
Exosomes were labeled by PKH26 Red Fluorescence Cell Linker Kit or PKH67 Fluorescent Cell Linker Kits (Sigma, St Louis, MO) following the manufacturer's instructions, SD rats (n=10) were injected with PBS and 100 mg PKH26 or PKH67-labeled MΦ exosomes through the tail vein.
After 24 h, EPCs were isolated from the spleen or PBL and analyzed by flow cytometry to quantify the proportion of cells that internalized MΦ exosomes.

Flow cytometry
To quantify the phenotypes of stimulated MΦ in each group, MΦ were stained using the following fluorochrome-labeled antibodies: anti-CD86-APC (BD, New Jersey, USA) and anti-CD206-FITC (BD). After staining for surface markers, MΦ were fixed and analyzed with an Accuri C6 flow cytometer (Becton Dickinson), and the results were analyzed using FlowJo software (Treestar, Inc.) (n=10).

Immunofluorescence
MΦ were seeded on glass coverslips in 6-well plates. After fixation and permeabilization, MΦ in each group were incubated with the primary antibody mouse-anti-CD86 (BD) overnight at 4°C and stained with secondary antibody Alexa Fluor 568 donkey anti-mouse IgG (Life Technologies) for 60 min at 18-24°C. Next, cells were again incubated with rabbit-anti-CD163 (BD) and Alexa Fluor 488 donkey anti-rabbit IgG (Life Technologies), followed by counter-staining of the nucleus 4 with Hoechst33342 dye (Sigma). Finally, the cells were imaged with a confocal imaging system (LSM780;Zeiss, GER) (n=10).
After fixation with 4% paraformaldehyde for 1 h, small-diameter TEBVs were cut longitudinally and fixed with double-sided tape on coverslips to expose the intima. Next, MΦ that were able to infiltrate small-diameter TEBVs were immunofluorescently stained as described above.
small-diameter TEBVs were transferred to another coverslip and imaged with the confocal imaging system (n=10).

ELISA
Cell culture supernatants and plasma of rats were used to detect the concentrations of inflammatory factors including IL-1β, IL-6, TNF-α, TGF-β, and IL-10 by ELISA kit (Boster Bioengineering Co. Ltd. China) following the manufacturer's instructions (n=10).

Immunoblotting
Exosomes and cells were lysed and protein content was assessed using BCA Protein Assay Kit (Biospes, Chongqing, China). Next, protein lysates were separated using SDS-PAGE, and

Co-culture experiments
For confocal microscopy, EPCs (1x10 4 ) were seeded on glass coverslips in the lower chamber of 12-well transwell plates. Absence (Control) or PKH26-stained MΦ (1x10 6 ) were added in the upper 5 chamber of a 0.4-μm pore membrane insert (Corning Transwell, New York, USA) for 24 h. Nuclei were stained with DAPI (blue) and cells were imaged with the confocal imaging system at various time intervals (n=10).

Nanoparticle trafficking analysis
Analysis of absolute size distribution of exosomes was performed using nanoparticle tracking analysis (NTA, Nano series, Malvern, UK). After isolation, the exosomes were diluted in 1 ml filtered PBS. Exosomes were measured at 23.75 ± 0.5°C and imaged at 25 frames per second for 60 s. Three detection times were performed for each sample (n=10).

Transmission electron microscopy (TEM)
For the morphology investigation by TEM, 5μl exosome pellet was placed on formvar carbon-coated 200-mesh copper electron microscopy grids, incubated, and subjected to standard uranyl acetate (Sigma) staining and then allowed to semi-dry at 18-24°C. MΦ were post-fixed with 1% osmium tetroxide (Sigma), progressively dehydrated in a graded ethanol series (30-100%), and embedded in epoxy resin. Thin (1-mm) and ultrathin (70-to 80-nm) sections were cut by a microtome (318423, Reichert Nr.) and placed on copper grids as described previously. [4] Next, the cells and exosomes were observed using a TEM (CM-10, Philips Netherlands). Micrographs were used to quantify the secreting process and diameter of exosomes (n=10).

Quantitative Reverse Transcription Polymerase Chain Reaction
Exosomal RNA was isolated using TRIzol LS Reagent (Invitrogen, Carlsbad, CA) and then reverse transcription polymerase chain reaction (qRT-PCR) was performed to measure miRNA and lncRNA expression following manufacturer's instruction. Relative expression was normalized to GAPDH (n=10). 6 EPCs cultured for 7 d were harvested and seeded into the upper chamber of a 24-well transwell migration insert (8μm; Corning). The lower chamber contained basic medium with or without 100 μg/ml exosomes derived from MΦ stimulated with LPS or netrin-1 in each group. After 12 h, the cells on the lower side were fixed and stained with crystal violet. Light microscopy was immediately used to observe the cell morphology and cell count (n=10).

EPCs migration, proliferation, and tube formation assay in vitro
EPCs cultured for 14 d were plated onto a 96-well plate (Costar, 1×10 4 cells per well) and incubated in each group as described above. The cells were incubated for 72 h and then 0.5 ml of 50 mM EdU (RiboBio Co., Ltd.) was added. After incubation for 6 h, EPCs were detected following the manufacturer's protocol (n=10).

Matrigel (BD) was added to 96-well plates (Corning) at 100μl/well and incubated for 30 min.
EPCs were then added to the wells and incubated in each group as described above. After 6, 12, and 24 h incubation, calcein (6.25μg/ml, Sigma) was added and EPCs tube formation was assessed by microscopy. The number of closed loops or meshes, branching points (nodes), tubes and total length of the tubes were calculated and quantified using MacBiophotonics ImageJ software (NIH) (n=10). [5]

Flow-chamber assessments
Platelets flow-chamber assessments were performed as described previously [6] with the following parameters: width, 16 mm; height, 5 mm; volume flow rate 0.2 ml/s, Q=0.103 ml/s. The circulating fluid (M199 medium enriched with 10% fetal calf serum) had a viscosity of μ=1.3 mPa/s, and the wall shear stress was τ=6Qμ/(wh2)=0.2 Pa. The suspending fluid density was 1.1 g/cm, and the mean linear flow rate was 12.9 mm/s, which produced a Reynolds number of 5.45. Circulation was maintained with a Peri-star Pro pump (WPI, USA) (n=10).

Small-diameter TEBVs construction and implantation
7 Under sterile conditions, carotid arteries were harvested from SD rats (200 g), rinsed with saline solution, and digested with 0.05% trypsin (Hyclone, Logan, UT, USA) at 37°C and under 5% CO 2 for 40 minutes to remove the cells. Next, nucleic acids were removed with RNase and DNase to obtain a vascular matrix consisting of collagen and elastic fibers. [7] The vascular matrix was incubated with 4 mg/ml collagen (Kensey Nash, Exton, PA, USA) for 24 h to crosslink a layer of collagen to both the internal and external surfaces of decellularized rat carotid arteries, and then incubated with nanoparticles of emulsified chitosan for 24 hours. Then 5 mM EDC was added for 24 h (to crosslink the collagen to the chitosan), and the vascular matrix material was incubated a second time with 4 mg/ml collagen for 24 h. Netrin-1-modified small-diameter TEBVs were prepared with nanoparticles that contained 500 ng/ml netrin-1 (R&D Systems), A2b-blocked small-diameter TEBVs were prepared with nanoparticles containing netrin-1 (500 ng/ml) and 100 μM MRS (Sigma). Netrin-1 was crosslinked to the matrix material by incubating the small-diameter TEBVs with 2 mg/ml of N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP, Pierce). [7][8][9] small-diameter TEBVs were transplanted into the carotid arteries of SD rats (200 g) after heparinization, anesthesia was provided with 10% chloral hydrate (Sigma), and the animals received intraperitoneal injections of heparin (1 mg/kg, Sigma) each day for 5 d after surgery (n=10).

Patency evaluation
The rats receiving small-diameter TEBVs transplantation were anesthetized, and the blood flow of the carotid artery was measured using Doppler flow analysis (Transonic System Inc T402). [10] Rats were injected with 1 ml heparin through the tail vein for 5 min, then their chests were opened and intravascular contrast agent iohexol (Tianheng Pharmaceutical Company, Nanoprobes, China) 8 was injected into the heart to evaluate the patency of the grafts in the micro-CT scanner (Skyscan1176, Bruker micro-CT, Kontich, Belgium). [11] After these tests, the transplanted small-diameter TEBVs were removed and SEM was used to examine intimal surface endothelialization. A portion of small-diameter TEBVs frozen sections were stained with H&E staining to examine patency and intimal hyperplasia (n=10).

Statistical analysis
Quantified data are reported as mean ± SD, and significance was evaluated using the Student's t-test.
Evaluations were performed with SPSS 13 software package, and a P value of less than 0.05 was considered significant.

Supplemental Video Legends
Video S1 A2b-blocked small-diameter TEBVs became occluded at 2 months after transplantation.
Video S3 Doppler vascular ultrasound revealed the patency of netrin-1-modified small-diameter TEBVs at 14 months after transplantation.