Sprague Dawley (SD) rats (male, weight 180-220 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Animals were maintained under controlled conditions with a 12/12 hours light/dark photoperiod, temperature of 22±3°C and humidity of 60±5%. This study was conducted with strict accordance to the Guide for the Care and Use of Laboratory Animals (Eighth Edition, 2011, published by The National Academies Press, 2101 Constitution Ave. NW, Washington, DC 20055, USA). The protocol was reviewed and approved by the Shanghai Ninth People’s Hospital Institutional Review Board (Permit Number: HKDL2013001b). Surgery was performed under sodium pentobarbital anesthesia with all efforts being made to minimize suffering.
2.2 Patient samples
A sample size of 30 patients with severe OSA and moderate ED (The International Index of Erectile Function: 5–12) admitted at Shanghai Ninth People’s Hospital, Shanghai, China was enrolled. According to hospital records, the patients had been clinically diagnosed with ED. The age of the patients ranged from 30 to 65 years and had a diagnosis of severe OSA, as verified by full-night attended polysomnography or polygraphy (i.e. apnea-hypopnea index ≥ 30 per hour of sleep). Patients with hypertension, diabetes, trauma, smoke, using drugs that affect erectile function, BMI＞35 and surgery history were excluded. Thirty healthy people (age-matched) to be used as controls were also recruited from the hospital. Serum samples were taken within 24 hours of symptom onset and frozen in liquid nitrogen and stored for short-term until further analyses. Ethical approval for the study was provided by the Independent Ethics Committee of Shanghai Ninth People’s Hospital, Shanghai, China. Guidelines from the Ethics Committee were followed where informed and written consent was obtained from all patients or their advisors before samples were collected.
2.3 Culturing ADSCs
Rats ADSCs were collected from the inguinal fat pad. Adipose tissues were washed with phosphate-buffered saline (PBS) to remove residual blood. The tissues were cut into 1mm2 pieces and digested in 1 mg/mL collagenase type II (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 1 h followed by centrifugation at 4000×g for 5 minutes. Obtained cell pellet was then suspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and 2 mmol/L L-glutamine. The cells were then cultured in a controlled environment having 5% CO2 and a temperature of 38°C for 48 hours. Cells were then transferred into fresh culture medium with subsequent subculture every 3 days. When cells were approximately 90% confluent, they were passaged and used at passage three. For Immunofluorescence: Cells were then incubated with conjugated monoclonal antibodies against CD29 (ab179471, 1:200), CD44 (ab189524, 1:200), CD90 (ab225, 1:200), CD105 (ab2529, 1:200) and vWF (ab194405, 1:200) (Abcam, Cambridge, UK) at 4 °C for 1 h to confirm the identity of ADSCs. Isotype-identical antibodies (#550343, 1:200, PharMingen) were used as controls. An Operetta High Content Imaging System (Perkin-Elmer, Waltham, MA, USA) was used to obtain the images of the cells. For Flow cytometer: Cells were identified and selected by flow cytometry (FCM) with anti-CD29, CD44, CD90, and CD105 (1:200; Abcam). After being subcultured to the third generation, cells at 80% confluence were washed twice with PBS followed by digestion with 0.25% trypsin–ethylenediaminetetraacetic acid (EDTA) (Thermo Fisher, MA, USA). The cells were then centrifuged at 1000 rpm and washed with PBS. After incubation with antibodies and their isotype controls (1:200) (PharMingen, CA, USA) at 4 °C for 30 min, the cells were flowed through the cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) at about 1000 cells per second and analysis.
2.4 Isolation of exosomes
Adipose-derived stromal cells collected from miR-301a-3p mimic (ADSCs transfected with miR-301a-3p overexpressing mimic), control (untreated ADSCs) and miR-NC (ADSCs transfected with miRNA mimic negative control) groups at 80%‐90% confluence were washed with PBS and cultured in microvascular endothelial cell growth medium-2 media deprived of FBS. ADSCs were then supplemented using 1×serum replacement solution (PeproTech) for 24 hours. Dead cells and debris were removed by centrifugation of ADSCs at 300×g for 10 minutes and 2000×g for 10 minutes followed by mixing 10 mL of the supernatant with 5 mL of ExoQuick-TC reagent (System Biosciences). The mixture was then centrifuged at 1500×g for 30 minutes, with the resulting exosome-containing pellet being re-suspended in nuclease-free water. TRIzol-LS (Invitrogen, CA, USA) and Exosomal Protein Extraction (Invitrogen) kits were used for extracting total RNA and protein, respectively. Isolated exosomes were used immediately for experiments or stored at -180°C. For transmission electron microscopy (TEM) observation: exosomes were stored in 1% paraformaldehyde, dehydrated via an ethanol series, and embedded in EPON. Sections (65 nm) were stained with uranyl acetate and Reynold’s lead citrate and examined with a transmission electron microscopy (CM-120 electron microscope, Philips). The specific exosome markers, including CD9, CD63, and TSG101 were identified by western blot analysis.
2.5 CIH exposure-induced ED rat model
Twenty-four male SD rats were randomly divided into control, CIH, CIH + exosomes from untreated ADSCs (Exo) and CIH + exosomes from miR-301a-3p overexpressing ADSCs (Exo-301a) groups (n = 6). An oxygen sensor was placed at the bottom of the chamber to measure the oxygen content in the CIH exposure chamber over the course of several cycles. Animals were exposed to 2 minutes of 5% O2 for each 4-minute cycle with each challenge lasting 8 hours. The challenge was done for eight weeks during the daytime from 8am to 4pm. Sham group rats were exposed to 21% O2. Exosomes (400 μg of protein) were isolated using 200 μL PBS and then administered using intracavernous injection for Exo groups, whereas control rats received an equal volume of PBS. Exosomes were administered to the rats every week for eight weeks.
2.6 Erectile function Measurement
After eight weeks of CIH exposure, the intracavernous pressure (ICP) and real-time carotid arterial pressure (RT-AP) were recorded simultaneously as described in our previous article. In brief, while under anesthesia, the right carotid artery, crus penis and bilateral cavernous nerves were exposed. Two 25-gauge catheters, filled with 250 U/ml heparin solution and connected to pressure transducer (Labchart, Colorado Springs, USA), were separately inserted into carotid artery and crus penis to record the RT-AP and ICP simultaneously. Using an electrode hook, we stimulated one side cavernous nerve at intervals of 5 minutes (3 times/per side). Stimulation parameters were 1.5 mA, 20 Hz, pulse width 0.2 ms, and duration 60s. The maximum ICP(MICP) and RT-AP of unilateral stimulation was selected for calculating mean ICP and mean AP of each rat. After stimulation, the penis was divided into two parts. One part was frozen in liquid nitrogen for western blot, another one was ﬁxed for histologic analysis.
Harvested tissues were immersed in optimal cutting temperature compound and immediately frozen in liquid nitrogen. The tissues were fixed in 4% paraformaldehyde, embedded in optimal cutting temperature and cut into sections having a thickness of 5μm followed by immunofluorescence staining as described in. Primary antibodies used in this study were eNOS (ab76198), nNOS (ab76067) and Phalloidin (ab176753) all obtained from Abcam at 37 °C for 2 h. Secondary antibodies included Alexa-488, Texas Red-conjugated antibodies, (1:500; Invitrogen, CA, USA) and Texas Red goat anti-rabbit IgG (1:200; Life Technologies, Grand Island, NY, USA). Nuclei were stained using 4',6-diamidino-2-phenylindole (DAPI) (1:10,000, Invitrogen, CA, USA). The number of cells in each image was counted with DAPI cells and positive cells. Ratio of positive nNOS counts to DAPI were used to analyze the nerve fibers in the dorsal section of the penis. Smooth muscle and endothelial stains were analyzed using the ratio of positively stained areas of phalloidin and eNOS to DAPI in the corpora cavernosa.
2.8 CCSMCs culture and CIH exposure
Rats were sacrificed where on a sterile table, the penis was excised and placed in a sterile Petri dish followed by two washes using PBS. The skin around the penis was carefully peeled away, along with the albuginea, urethral sponge, cavernous body, and other vessels. The corpus cavernosum was cut into 1mm3 tissue blocks that were placed in a cell culture flask containing 0.5% type I collagenase solution (Sigma). Cells were cultured at 37°C with shaking in a humidified atmosphere having 95% air and 5% CO2 for 3 hours. The cells were then filtered and centrifuged followed by the addition of 3ml F12 medium (Invitrogen) containing 20% fetal bovine serum (Invitrogen) and incubated at 37°C and 5% CO2. Long, spindle-shaped SMCs were observed at the bottom of the 25cm2 culture flasks after incubating for 24 hours. For CIH exposure, CCSMCs were exposed to 5min of 14% to 15% O2 during each 60min cycle for 24 hours by using BioSpherix-OxyCycler C42system (BioSpherix, Redfield, NY). All cells were cultured for 24 hours followed by co-culturing with miR-301a-3p-enriched exosomes for 48 hours.
2.9 Statistical analysis
Results are expressed as the mean ± SD. All the data obtained from this study was analyzed using GraphPad 9.0. Two groups analysis was performed t-test (two tailed). One-way ANOVA was used among various groups with p < 0.05 being considered as statistically significant.
More detailed materials and methods are in the Supplementary Methods.