MicroRNA-148a-3p in pericyte-derived extracellular vesicles improves erectile function in diabetic mice by promoting cavernous neurovascular regeneration

This animal model study included 75 adult male C57BL/6 J mice (8 weeks old; Orient Bio, Inc.): 15 for the MCPs primary culture, MCPs-EVs isolation, and small-RNA sequencing analysis; 20 for in vitro studies; and 40 for in vivo studies. All animal experiments were approved by the Ethics Committee at the Inha University (approval number: INHA 200309-691). Animals were monitored daily for health and behavior as previously described [14, 15]. In all studies, animals were anesthetized with an intramuscular injection of ketamine (100 mg/kg; Yuhan Corp., Seoul, Korea) and xylazine (5 mg/kg; Bayer Korea, Seoul, Korea). Animals were euthanized by 100% CO₂ gas replacement; cessation of heartbeat and respiration were confirmed prior to harvesting tissue. All animal studies were randomized and blinded. No mice died during the experimental procedures.

Primary MCPs and MCECs were prepared from mouse penile tissue as previously described [16, 17]. Briefly, 8 weeks old male C57BL/6 J mice were euthanized by 100% CO₂ gas replacement. Then, the penis tissues were harvested and maintained in sterile vials with HBSS (Gibco). After washing with PBS for 3 times, the urethra and dorsal neurovascular bundle were removed, and only the corpus cavernosum tissues were used. For MCPs culture, 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. Then, 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. For MCECs culture, corpus cavernosum tissues were cut into approximately 1-2 mm sections, put into 60 mm dishes, and covered by Matrigel (Becton Dickinson, Mountain View, CA, USA). Tissues were cultured with the complement M199 medium (Gibco) containing 20% fetal bovine serum (FBS, Gibco), 0.5 mg/mL of heparin (Sigma-Aldrich), 5 ng/mL of recombinant human vascular endothelial growth factor (VEGF, R&D Systems Inc., Minneapolis, MN, USA), and 1% penicillin/streptomycin (Gibco) in a 5% CO2 atmosphere incubator at 37 °C. After cells were confluent on the bottom of the 60 mm cell culture dishes (approximately 2 weeks of culture), sprouting cells were sub-cultured into other cell culture dishes coated with 0.2% gelatin (Sigma-Aldrich, St Louis, MO, USA). Cells from passages 2 to 3 were used for the experiments. Diabetes-induced angiopathy was mimicked by serum-starving cells (MCECs and MCPs) for 24 hours and then exposing them to normal-glucose (NG; 5 mM glucose, Sigma-Aldrich, St. Louis, MO, USA) or high-glucose (HG; 30 mM glucose) conditions for 3 days [18].

MCPs-EVs were isolated from DMEM-cultured MCPs (Gibco; Carlsbad, CA, USA) in medium supplemented with 20% exosome-depleted FBS (Gibco), with 1% penicillin/streptomycin (Gibco), using a commercial EV isolation kit (ExoQuick-TC, System Biosciences, LLC., Palo Alto, CA, USA) according to the manufacturers’ instructions. The EXOCET exosome quantitation kit (System Biosciences, LLC.) was used to quantify MCPs-EVs, and their concentration was adjusted to 1 μg/μL before treatments.

The MCPs-EV morphology was ascertained by transmission electron microscopy (TEM; Electron Microscopy Sciences, Fort Washington, PA, USA) and their characterization was validated based on the expression of one negative and three positive EV markers in Western blotting [11] (negative marker: GM130 [1:1000; BD Biosciences, San Jose, CA, USA]; Three positive EV markers [1:1000]: CD9 [Abcam, Cambridge, MA, USA), CD63 [NOVUS Biologicals, Littleton, CO, USA], and CD81 [NOVUS Biologicals]).

MCPs-EVs were labelled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) red-fluorescent dye (ThermoFisher Scientific, Inc., Carlsbad, CA, USA) according to the manufacturer’s instructions. After 6-hour DiD-labeled MCPs-EV treatment, MCECs were fixed by incubation with 4% formaldehyde for 15 minutes. DiD dye tracking was determined under a confocal fluorescence microscope (K1-Fluo, Nanoscope Systems, Inc., Daejeon, Korea).

The small-RNA-sequencing assay was performed by E-Biogen Inc. (Korea). For control and test RNAs, a library was constructed using the NEBNext Multiplex Small RNA Library Prep kit (New England BioLabs, Inc., USA) according to the manufacturer’ instructions. The small-RNA-sequencing data were deposited in the Gene Expression Omnibus database (www.​ncbi.​nlm.​nih.​gov/​geo; accession no. GSE195533).

Primary MCPs were transfected with 20 nM miR148a-3p inhibitor (a mirVana® miRNA inhibitor, Cat #, 4,464,084, Thermo Fisher, San Jose, CA, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 24 hours, the culture medium was replaced with DMEM supplemented with 20% exosome-depleted FBS with 1% penicillin/streptomycin for 3 days, and the culture medium was collected for conditioned MCPs-EV isolation.

Exosomal RNA was isolated from 150 ul of MCPs-EVs using the miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol as described previously [19].

Total RNA from MCPs was isolated using TRIzol (Invitrogen) according to the manufacturer’s instructions. miRNAs were reverse-transcribed using the Taqman microRNA Reverse Transcription kit (Applied Biosystems, Carlsbad, CA, USA) and the associated miRNA-specific stem-loop primers (Applied Biosystems). The RT reaction conditions were: 30 minutes at 16 °C to anneal primers, 30 minutes at 42 °C for extension of primers on miRNA and synthesis of the first cDNA strands, and 5 minutes at 85 °C to stop the reaction. qPCR was performed on the 7500 Fast Real-Time PCR System (Applied Biosystems) with the following conditions: one denaturing step at 95 °C for 10 minutes, followed by 40 denaturing cycles at 95 °C for 15 seconds; and annealing and elongation at 60 °C for 60 seconds. The data presented correspond to the mean of 2^-ddct from at least three independent experiments after normalization to U6.

The nitrite assay kit (MAK367, Sigma-Aldrich) was used to determine nitric oxide (NO) concentration in MCECs, according to the manufacturer’s protocol as described previously [14]. MCECs were seeded in 6-well plates at a density of 5 × 10⁵ cells/well in 2 mL of M199 medium. After 24 hours, MCECs were exposed to glucose conditions with or without MCPs-EV or miR-148a-3p-depleted MCPs-EV for 72 hours at 37 °C in a humidified 5% CO₂ atmosphere. Then, cultured medium was collected for NO concentration measurement. Nitrite levels was measured at a wavelength of 540 nm, using a microplate spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA). Each experiment contained six replicates and repeated four times.

Tube-formation assays were performed as described previously [20]. Tube formation was monitored for 18 hours under a phase-contrast microscope (CKX41, Olympus, Tokyo, Japan). The number of master junctions from four separate experiments were quantified using Image J (National Institutes of Health [NIH] 1.34, http://​rsbweb.​nih.​gov/​ij/​).

The migration assay was performed with the SPLScar™Block system (SPL life sciences, Pocheon-si, Gyeonggi-do, Korea) on 60-mm culture dishes [20]. Images were obtained using a phase-contrast microscope (Olympus), and cell migration was analyzed by determining the ratio of cells that moved into the frame line in the figures from four separate block systems using Image J.

The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay was performed using the ApopTag® Fluorescein In Situ Apoptosis Detection Kit (Chemicon, Temecula, CA, USA) as described previously [21]. Samples were mounted in a solution (Vector Laboratories Inc., Burlingame, CA, USA) containing 4,6-diamidino-2-phenylindole (DAPI), a nuclear stain. Digital images were obtained using a confocal fluorescence microscope (K1-Fluo; Nanoscope Systems, Inc). The number of apoptotic cells was counted using Image J.

Diabetes was induced in 8-week-old C57BL/6 J mice by injecting them with streptozotocin (STZ; 50 mg/kg, Sigma-Aldrich) for 5 consecutive days [22]. After 8 weeks, mice were randomly distributed into four groups: control nondiabetic mice and STZ-induced diabetic mice receiving two successive intracavernous injections of PBS (20 μL; days − 3 and 0), two successive intracavernous injections of MCPs-EV control reagent (5 μg in 20 μL PBS, days − 3 and 0), or two successive intracavernous injection of miR-148a-3p-depleted MCPs-EV (5 μg in 20 μL PBS, days − 3 and 0) into the midportion of the corpus cavernosum (n = 5 per group). For injections, mice were anesthetized with intramuscular injections of ketamine (100 mg/kg) and xylazine (5 mg/kg) and positioned supine on a thermoregulated surgical table and the base of the penis was compressed with a vascular clamp before injection. After injection, the clamp was left in place for 30 minutes to prevent backflow of blood from the penis.

Two weeks later, the erectile function measurement was performed as previously described [22]. Systemic blood pressure was measured continuously by using a noninvasive tail-cuff system (Visitech Systems, Apex, NC, USA) before the measurement of intracavernous pressure (ICP). The ratios of maximal ICP and total ICP to mean systolic blood pressure (MSBP) were calculated to normalize for variations in systemic blood pressure.

For fluorescence microscopy, mice penile tissue was fixed in 4% paraformaldehyde for 24 hours at 4 °C, and the frozen tissue sections (12-μm thick) were incubated overnight at 4 °C with primary antibodies including: CD31 (endothelial cell marker, 1:50; Millipore, Temecula, CA, USA), NG2 antibody (pericyte marker, 1:50; Millipore), β (III)-tubulin antibody (neuronal cell marker, 1:200; Abcam), nNOS (neuronal cell marker, 1:100; Santa Cruz Biotechnology Inc., Dallas, TX USA), or phospho-Histone H3 (PH3; Mitosis marker, Upstate Biotechnology Inc., Temecula, CA, USA). After several washes with PBS, samples were incubated with donkey anti-rabbit DyLight® 550 (1:200; Abcam), goat anti-Armenian hamster Fluorescein isothiocyanate (FITC; 1:200; Jackson ImmunoResearch Laboratories, West grove, PA, USA), donkey anti-rabbit FITC (1:200; Jackson ImmunoResearch Laboratories), and donkey anti-chicken Tetramethylrhodamine (TRITC) secondary antibodies (1:200; Jackson ImmunoResearch Laboratories) for 2 hours at room temperature. Using a DAPI-based solution (Vector Laboratories Inc.), samples were mounted for nuclear staining. Samples were visualized and images were obtained with a confocal microscope (Nanoscope Systems, Inc). Quantitative analysis was performed using Image J.

Computational predictions of miRNA target genes were obtained using the following published algorithms: DIANA-microT-CDS (http://​www.​microrna.​gr/​microT-CDS), TargetScan (http://​www.​targetscan.​org), and miRDB (http://​www.​mirdb.​org).

All results are expressed as the mean ± SEM of at least four independent experiments. The unpaired t-test was used to compare two groups, and one-way ANOVA followed by Tukey’s post hoc test was used for four-group comparisons. The analysis was conducted using GraphPad Prism version 8 (Graph Pad Software, Inc.). P values less than 0.05 were considered statistically significant. Statistical sample sizes were determined based on our previous studies [15, 23]. In vivo functional evaluation requires at least 5 animals per group, and other experiments require at least 4 samples per group for more effective statistical analysis.

Based on the TEM images, the MCPs-EVs exhibited a unique cup-shaped morphology (diameter ~ 30 nm; Fig. 1a). Western blotting showed that positive EVs surface markers, such as CD9, CD63, and CD81, were present in MCPs-EVs but had low expression levels in MCP lysate. Conversely, negative EV surface markers, such as GM130, were not detected in purified MCPs-EVs (Fig. 1b). To determine whether the secreted MCPs-EVs were taken up by endothelial cells, DiD red dye-labeled MCPs-EVs were treated in MCECs and were detected inside the MCECs after 6 hours (Fig. 1c). These data suggest that MPCs-EVs are taken up by MCECs.

To assess the composition of miRNAs in MCPs-EVs, we performed small-RNA sequencing analysis with MCPs-EVs. We detected only 90 miRNAs in MCPs-EVs, and the top 10 expressed miRNAs are listed in Table 1. We focused on miR-148a-3p, the most abundant miRNA in MCPs-EVs.

To assess the angiogenic effect of miR-148a-3p, which has the highest expression in MCPs-EVs, we transfected a miR-148a-3p inhibitor in MCPs and isolated MCPs-EVs, we found that the expression of miR-148a-3p was significantly reduced in MCPs and MCPs-EVs (Fig. 2a and b). Then, we evaluated the expression of miR-148a-3p and nitrite levels in MCECs, which was treated with PBS, MCPs-EVs-regent control (MCPs-EVs-RC), MCPs-EVs-miR-148a-3p inhibitor (MCPs-EVs-miR-148a-3p-i) under normal-glucose (NG) and high-glucose (HG) conditions. We found that the expression of miR-148a-3p (Fig. 2c) and nitrite levels (Fig. 2d) were significant reduced in MCECs under HG conditions, and MCPs-EVs treatment leads to the recovery of miR-148a-3p expression and nitrite levels. However, there was no significant change in the expression of miR-148a-3p and nitrite levels in the group treated with miR-148a-3p-depleted MCP-EVs under HG conditions. Next, tube-formation (Fig. 2e) and cell-migration (Fig. 2f) abilities were significantly reduced in MCECs exposed to the high-glucose PBS environment. Reagent-controlled MCPs-EVs can induce tube formation and migration of endothelial cells under high-glucose conditions, thereby promoting angiogenesis. However, these effects were significantly attenuated in the group treated with miR-148a-3p-depleted MCPs-EVs under high-glucose conditions (Fig. 2e–h). Furthermore, using TUNEL assay and PH3 staining, we found that miR-148a-3p-depleted MCPs-EVs were unable to reduce apoptosis (Fig. 3)a-b or induce proliferation (Fig. 3c and d) of MCECs under high-glucose conditions. Collectively, these results suggest that miR-148a-3p plays an important role in MCPs-EV-promoted angiogenesis by inducing MCEC migration, survival, and proliferation under high-glucose conditions.

To investigate whether MCPs-EVs have beneficial effects via miR-148a-3p on erectile function in diabetic mice, we intracavernously injected the reagent-controlled or miR-148a-3p-depleted MCPs-EVs and evaluated erectile function 2 weeks later. During electrical stimulation, the ratios of maximal and total intracavernous pressure (ICP) to MSBP were significantly lower in PBS-treated diabetic mice than in age-matched non-diabetic controls. Interestingly, diabetic mice treated with MCPs-EVs had significantly improved erection parameters, reaching almost 93% of the values in controls. However, miR-148a-3p-depleted MCPs-EV treatment showed no such effects in diabetic mice (Fig. 4). Immunofluorescence staining for CD31 (Fig. 5a, c), NG2 (Fig. 5 a, d) in cavernosum tissues and β (III)-tubulin (Fig. 5 b, e) and neuronal NOS (nNOS; Fig. 5 b, f) in dorsal nerve bundles demonstrated that MCPs-EVs significantly improved the endothelial cell, pericyte, and nerve composition in diabetic mice. Fasting and postprandial blood glucose concentrations were significantly higher in diabetic mice than in control mice. However, there were no significant differences in body weight and blood glucose levels in diabetic mice regardless of treatment (Table 2). No detectable differences in MSBP were observed between the three STZ-induced experimental groups. These results suggest that miR-148a-3p plays an important role in MCPs-EVs-induced improvement in erectile function by rescuing cavernous neurovascular regeneration in diabetic mice.

There were 98 target genes for miR-148a-3p that were predicted from the DIANA-microT-CDS, TargetScan, and miRDB according to the following parameters: target score > 80, intersection in all three algorithms, and are detected in MCECs through the published RNA-sequencing result [20]. From the literature review and removal of genes to identify a target of miR-148a-3p, we selected 5 predicted target genes: Ago2, S1pr1, PDK4, Prkaa1, and Adam10. Of these genes, we focused on PDK4 expression which was significantly regulated by miR-148a-3p (Fig. 6a, b). Using TargetScan, the binding sequences of miR-148a-3p at position 655-662 of PDK4 3’UTR is shown in Fig. 6c. The luciferase reporter assay demonstrated that luciferase activity was significantly reduced in the PDK4 3’UTR plasmid and miR148a-3p-mimic co-transfected group. However, the luciferase activity in the control vector-transfected group showed no difference (Fig. 6d). These results suggest that miR-148a-3p inhibits PDK4 expression by directly binding to the 3’UTR of PDK4 mRNA.