RNA-Sequencing Profiling Analysis of Pericyte-Derived Extracellular Vesicle-Mimetic Nanovesicles-Regulated Genes in Primary Cultured Fibroblasts From Normal and Peyronie’s Disease Penile Tunica Albuginea


 BackgroundPeyronie’s disease (PD) is a severe fibrotic disease of the tunica albuginea that causes penis curvature and leads to penile pain, deformity, and erectile dysfunction. The role of pericytes in the pathogenesis of fibrosis has recently been determined. Extracellular vesicle (EV)-mimetic nanovesicles (NVs) have attracted attention regarding intercellular communication between cells in the field of fibrosis. However, the global gene expression of pericyte-derived EV-mimetic NVs (PC-NVs) in regulating fibrosis remains unknown. Here, we used RNA-sequencing technology to investigate the potential target genes regulated by PC-NVs in primary fibroblasts derived from human PD plaque. MethodsHuman primary fibroblasts derived from normal and PD patients was cultured and treated with cavernosum pericytes isolated extracellular vesicle (EV)-mimetic nanovesicles (NVs). A global gene expression RNA-sequencing assay was performed on normal fibroblasts, PD fibroblasts, and PD fibroblasts treated with PC-NVs. Reverse transcription polymerase chain reaction (RT-PCR) was used for sequencing data validation. ResultsA total of 4135 genes showed significantly differential expression in the normal fibroblasts, PD fibroblasts, and PD fibroblasts treated with PC-NVs. However, only 91 contra-regulated genes were detected among the three libraries. Furthermore, 20 contra-regulated genes were selected and 11 showed consistent changes in the RNA-sequencing assay, which were validated by RT-PCR. ConclusionThe gene expression profiling results suggested that these validated genes may be good targets for understanding potential mechanisms and conducting molecular studies into PD.

detected among the three libraries. Furthermore, 20 contra-regulated genes were selected and 11 showed consistent changes in the RNA-sequencing assay, which were validated by RT-PCR.

Conclusion
The gene expression pro ling results suggested that these validated genes may be good targets for understanding potential mechanisms and conducting molecular studies into PD.

Background
Peyronie's disease (PD) is caused by excessive brosis and scar tissue formation in the tunica albuginea (TA), resulting in penile pain, abnormal curvature, and erectile dysfunction (ED) [1,2]. Although the existence of PD has been known for a long time, the pathophysiology of PD has not been studied as widely as brosis in other organs, such as the kidneys, liver, or lungs. Currently, the most available medical therapy is collagenase and interferon injection and surgical intervention [3,4]. However, these treatments can cause glandular hypoesthesia and a high risk of new onset ED [5]. Therefore, the identi cation of novel therapeutic targets related to PD brosis is required.
Pericytes play a fundamental role in vascular contractility and stability, regulation of vascular development, and as a storage vault of mesenchymal stem cells [6,7]. In vitro studies have shown that pericytes exhibit brogenic potential [8,9] and transition to myo broblasts [10]. Moreover, the inhibition of angiogenesis may be effective in the suppression of brosis [11]. However, recently studies have shown that the inhibition of angiogenesis may aggravate brosis [12,13]. These ndings suggest that different antiangiogenic and molecular targets produce different results in the treatment of brosis.
Extracellular vesicles (EVs) were previously believed to be cell excretions. However, a number of studies have shown that EVs contain proteins, lipids, and RNA, which can affect the physiological and pathological communications between cells [14,15]. Many studies regarding the potential role of EVs have been conducted for human diseases, including strokes [16], tumor metastasis [17], and kidney disease [18]. Therefore, clarifying the role of EVs in brosis would be bene cial to aid in the understanding of brosis mechanisms. However, one of the major limitations of EVs is the low production yield [19]. Therefore, to maximize the production of vesicles, we used a mini extruder system and extracted more than 100-fold greater EV-mimetic NVs from pericytes. The cell-derived EV-mimetic NVs showed similar characteristics to the natural EVs [20]. These studies suggest that pericyte-derived EV-mimetic NVs (PC-NVs) may be bene cial for the functional study of brosis.
Gene expression pro ling analysis in physiological and pathological conditions can provide a foundation for studying the mechanisms of brosis in PD. In the present study, we performed an RNA-sequencing assay on normal broblasts, PD broblasts, and PD broblasts treated with PC-NVs.

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 broblast 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 broblasts
The TA tissues were used for the primary broblast culture as described previously [21,22]. Brie y, 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 modi ed 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% CO 2 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% CO 2 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.

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 sacri ced 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% CO 2 atmosphere. Thereafter, 900 µL of complement medium was added and incubated at 37°C with 5% CO 2. 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.

RNA-sequencing assay
For the RNA-sequencing study, the normal and PD TA-derived broblasts were cultured and treated with PC-NVs (n = 4 per group). The RNA-sequencing assay was performed by E-Biogen Inc. (Korea). Brie y, 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 quanti cation 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. Quanti cation was performed using the library quanti cation 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).
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].

Statistical analysis
All data are expressed as means ± standard errors. Statistical analysis was performed using Student ttest. P values less than 0.05 were considered statistically signi cant.

Identi cation of human fibroblasts
The broblasts were isolated from human normal and PD plaque tissues. Representative images showed high positive staining for CD90 and Vimentin ( broblast markers) of more than 95%, but not for pericyte (NG2) or endothelial cell (CD31) markers (Fig. 1a, b).

PC-NV preparation and characterization
PC-NVs were prepared from MCPs according to previous methods [26]. Western blot analysis showed that PC-NVs displayed positive exosomes markers, including CD9, CD81, and TSG101, but not for negative marker GM130 (Fig. 1c, d).

Transcriptional pro ling and gene ontology (GO) category analysis
For this study, three gene libraries for the normal broblast (NF), PD broblast (PF), and PC-NVs-treated PF (PFPC) groups were constructed for an RNA-sequencing assay (n = 4 for each group). In total, 25737 genes were detected in three libraries. Signi cant gene selection was performed with three conditions: fold-change > 2.0, log2 > 4, and p-value < 0.05. Among all detected genes, 3961 showed signi cant differential expression in the PF group compared with the NF group, and 174 were signi cantly differentially expressed in the PFPC group compared with the PF group (Fig. 2a, b, c). Only 91 contraregulated genes (Supplementary Table S1) were detected between PF/NF and PFPC/PF through Venn diagram analysis (Fig. 2d).

Validation of RNA-sequencing results by RT-PCR
To validate the RNA-sequencing results, we selected 20 genes (Supplementary Table S2) from 91 contraregulated DEGs, and 11 (primers as shown in Supplementary Table S3) showed results consistent with the RNA-sequencing assay by RT-PCR. Among these genes, MMP3, AKR1C1, SMOC1, ANGPTL2, SEMA3A, TRIM15, EGR1, and BMP2 were downregulated in the PF group compared with the NF group, and were signi cantly recovered in the PFPC group (Fig. 4a, c). Only TFPI2, SFRP4, and SERPINE1 were induced in the PF group and recovered in the PFPC group (Fig. 4b, d).

Discussion
The accurate physiological and pathological mechanisms of PD remain poorly understood. To date, most gene expression studies have focused on human PD plaque at a tissue level in vivo [32,33] but not at a cellular level in vitro. Therefore, to investigate the exact mechanisms and potential target genes for PD, we cultured human broblasts from human PD plaque and performed RNA-sequencing assays.
EVs display a potential role in kidney brosis and other brotic diseases [18,34]; however, little is currently known regarding the detailed mechanisms. Considering the low yield of EVs, we extracted more than a 100-fold greater EV-mimetic NVs from MCPs, which were primarily cultured from mouse corpus cavernosum tissues. Many previous studies have found that pericytes display diverse features in relation to brosis that are dependent on different molecular targets [8,9,11]. In this study, human PD broblasts exposed to PC-NVs were compared with human PD broblasts to investigate the regulation of gene expression by PC-NVs in PD.
From the RNA-sequencing assay, 3961 DEGs were detected, and the 16 top GO categories were assessed in this study. GO analysis showed that signi cantly altered genes were enriched in the extracellular matrix, angiogenesis, and brosis. The extracellular matrix is a driver of progressive brosis [35], and angiogenesis is closely associated with chronic liver brosis [18]. These data suggest that our DEG detection is credible. However, the molecular basis of PC-NVs in regulating the extracellular matrix or angiogenesis pathway in PD remains largely unknown. In this study, only 91 contra-regulated genes were identi ed from the three libraries (NF, PF, and PFPC). After precision screening, 20 genes were selected and validated by RT-PCR in same conditions. However, only 11 genes were validated to be consistent with the RNA-sequencing results. These genes may be the key to understanding how PC-NVs regulate the extracellular matrix, angiogenesis, and brosis mechanisms in PD.
To the best of our knowledge, this is the rst study to demonstrate the systematic pro ling of gene alterations in NF, PF, and PFPC. However, the present study has some limitations. First, a small number of cultured human broblast samples were used in target gene validation and age differences existed among groups. Second, we were unable to demonstrate the network of these validated genes in the extracellular matrix, angiogenesis, and brosis pathways. Third, mouse corpus cavernous pericytes were used for EV-mimetic NVs isolation, and further studies are required to evaluate the role of human corpus cavernous pericytes isolated with EV-mimetic NVs in PD.

Conclusion
In summary, we pro led the DEGs of human TA cultured broblasts in NF, PF, and PFPC groups. We hypothesize that these validated genes are good candidates for the study of the mechanism of PC-NVs in PD. Further studies exploring the effect of these target genes will be bene cial to further our understanding of the detailed mechanisms of the extracellular matrix, angiogenesis, and brosis in PD.