Ethics statement and Study design
All TA tissues and animals used in this study were approved by the Institutional Review Board (IRB No: 2007-730) and the Institutional Animal Care and Use Committee of our University (approval number: 171129-527), respectively. The plaque tissue of a patient with PD (48 years old) and the normal TA tissue from control patients (undergoing penoplasty for congenital curvature, 21 years old) were used for the human fibroblast culture study. In addition, 10 adult male C57BL/6J mice (8 weeks old, Orient Bio, Korea) were used for the mouse cavernous pericytes (MCPs) primary culture.
Primary culture and characterization of human fibroblasts
The TA tissues were used for the primary fibroblast culture as described previously [21, 22]. Briefly, PD plaque and normal TA tissues were maintained in sterile vials with Hank’s balanced salt solution (HBSS, Gibco, Carlsbad, CA, USA) and washed three times with phosphate-buffered saline (PBS). The TA tissues were cut into 1-2 mm sections and incubated in 12.5 mL Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 0.06% collagenase A (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 1 h in a 5% CO2 atmosphere. The cells and tissue fragments were collected by centrifugation (400 g for 5 min), washed with fresh culture medium, and placed in 100 mm cell culture dishes (Falcon-Becton Dickinson Labware, Franklin Lakes, NJ, USA) with DMEM containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37°C in a 5% CO2 atmosphere. Media were changed every 2 days and the cells were characterized as previously described [21, 22]. Passages 5 to 8 were used for the experiments.
To determine cell type, the cells were cultured on sterile cover glasses, placed into 12-well plates and grown until nearly confluent. The cells were stained with antibody to CD90 (fibroblast marker, R&D Systems Inc., Minneapolis, MN, USA; 1:100), Vimentin (fibroblast marker, R&D Systems Inc., 1:50), NG2 chondroitin sulfate proteoglycan (NG2, pericyte marker, Millipore, San Francisco, CA, USA; 1:50), CD31 (endothelial cell marker, Chemicon, Temecula, CA, USA; 1:50), or DAPI (nucleus marker; Vector Laboratories Inc., Burlingame, CA, USA), as previously described [23]. Signals were visualized and digital images were obtained using a confocal fluorescence microscope (K1-Fluo, Nanoscope Systems, Inc., Daejeon, Korea).
Primary culture of MCPs
The primary cultures of MCPs were performed as described previously [24, 25]. Shortly, 8 weeks old male C57BL/6J mice were anesthetized with ketamine (100 mg/kg) and xylazine (5 mg/kg) intramuscularly, and sacrificed by cervical dislocation. Then, the penis tissues were harvested and maintained in sterile vials with HBSS (Gibco). After washing three times with PBS, the urethra and dorsal neurovascular bundle were removed, and only the corpus cavernosum tissues were used. The corpus cavernosum tissues were cut into approximately 1-2 mm sections and settled via gravity into collagen I-coated 35 mm cell culture dishes with 300 µL complement DMEM (GIBCO) at 37°C for 20 min in a 5% CO2 atmosphere. Thereafter, 900 µL of complement medium was added and incubated at 37°C with 5% CO2. The complement medium contained 20% FBS, 1% penicillin/streptomycin, and 10 nM human pigment epithelium-derived factor (PEDF; Sigma-Aldrich). The medium was changed every 2 days, and after approximately 10 days sprouting cells were sub-cultured into collagen I (Advanced BioMatrix, San Diego, CA, USA)-coated dishes. Cells from passages 2 to 3 were used for the experiments.
Preparation and characterization of MCP-derived EV-mimetic nanovesicles (NVs)
MCP-derived EV-mimetic NVs (PC-NVs) were prepared using a mini extruder system (Avanti Polar Lipids, Birmingham, AL, USA), as described previously [26, 27]. Briefly, MCPs were washed three times with PBS, detached with 0.25% Trypsin-EDTA (Invitrogen, Carlsbad, CA, USA) and re-suspended in 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer solution (Gibco). The cell suspension was sequentially extruded 10 times across 10, 5, and 1 μm pore-sized polycarbonate membranes (Nuclepore, Whatman Inc., Clifon, NJ, USA), respectively. Next, ultracentrifugation was performed at 100,000 g for 2 h at 4°C with a step gradient, which was formed with 50% iodixanol (1 mL; Axis-Shield PoC AS, Oslo, Norway) overlaid with 10% iodixanol (2 mL) and topside with the extruded samples (7 mL). PC-NVs were filtered with a 0.45 μm filter and stored at -80°C until further use. To quantify the PC-NVs, the EXOCET exosome quantitation assay kit (System Biosciences, Palo Alto, CA, USA) was used, and 1 µg/µL of the final concentration of the PC-NVs was prepared for all experiments.
Western blotting
For the immunoblot analyses of PC-NVs, equal protein amounts (10 µg) of purified PC-NVs and whole cells extracted using RIPA lysis buffer (Sigma-Aldrich) were separated by SDS-PAGE (12% gel) and transferred to polyvinylidene fluoride (PVDF) membranes. Each blot was blocked and incubated with antibodies to GM130 (BD Biosciences, San Jose, CA, USA; 1:1000), CD9 (Abcam, Cambridge, UK; 1:1000), CD81 (Novus Biologicals; 1:1000), or TSG101 (Novus Biologicals; 1:500).
RNA-sequencing assay
For the RNA-sequencing study, the normal and PD TA-derived fibroblasts were cultured and treated with PC-NVs (n = 4 per group). The RNA-sequencing assay was performed by E-Biogen Inc. (Korea). Briefly, total RNA was isolated 24 h after exposure to PC-NVs using TRIzol reagent (Invitrogen). RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Amstelveen, The Netherlands), and RNA quantification was performed using an ND-2000 Spectrophotometer (Thermo Inc., DE, USA).
Library sequencing and data analysis
Libraries were prepared from total RNA using the SMARTer Stranded RNA-Seq Kit (Clontech Laboratories, Inc., USA). The isolation of mRNA was performed using the Poly(A) RNA Selection Kit (LEXOGEN, Inc., Austria). Indexing was performed using the Illumina indices 1-12. The enrichment step was performed using PCR. Subsequently, libraries were checked using the Agilent 2100 Bioanalyzer (DNA High Sensitivity Kit) to evaluate the mean fragment size. Quantification was performed using the library quantification kit using a StepOne Real-Time PCR System (Life Technologies, Inc., USA). High-throughput sequencing was performed as paired-end 100 sequencing using HiSeq 2500 (Illumina, Inc., USA).
Quality control of the raw sequencing data was performed using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Adapter and low-quality reads (<Q20) were removed using FASTX_Trimmer (http://hannonlab.cshl.edu/fastx_toolkit/) and BBMap (https://sourceforge.net/projects/bbmap/). Then, the trimmed reads were mapped to the reference genome using TopHat [28]. Gene expression levels were estimated using RC (read count) and FPKM (fragments per kb per million reads) values by BEDTools [29] and Cufflinks [30]. The expression values were normalized with the Quantile normalization method using EdgeR within R (https://www.r-project.org). Data mining and graphic visualization were performed using ExDEGA (E-Biogen, Inc., Korea). The RNA-sequencing data have been deposited in the Gene Expression Omnibus database (www.ncbi.nlm.nih.gov/geo accession no. GSE146500).
Validation of sequencing data by RT-PCR
Total RNA was extracted from cultured cells using TRIzol (Invitrogen) following the manufacturer’s protocols. Reverse transcription was performed using 1 µg of RNA in 20 µL of reaction buffer with oligo dT primer and AccuPower RT Premix (Bioneer Inc., Korea). The PCR reaction was performed with denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min in a DNA Engine Tetrad Peltier Thermal Cycler. For the analysis of PCR products, 10 µL of each PCR product was electrophoresed on a 1% agarose gel and detected under ultraviolet light. GAPDH was used as an internal control [31].
All digital image, western blot, and PCR band densitometry analyses were performed using an image analyzer system (National Institutes of Health [NIH] ImageJ 1.34, http://rsbweb.nih.gov/ij/).
Statistical analysis
All data are expressed as means ± standard errors. Statistical analysis was performed using Student t-test. P values less than 0.05 were considered statistically significant.