Knockdown of miR‐423‐5p simultaneously upgrades the eNOS and VEGFa pathways in ADSCs and improves erectile function in diabetic rats

Abstract This study aimed to explore the possibility of miR‐423‐5p modified adipose‐derived stem cell (ADSCs) therapy on streptozotocin (STZ)‐induced diabetes mellitus erectile dysfunction (DMED) rats. MiR‐423‐5p was knocked down in ADSCs. ADSCs, NC‐miR‐ADSCs and miR‐ADSCs were co‐cultured with human umbilical vein endothelial cells (HUVECs). Normal and high glucose media were supplemented. The supernatant and HUVECs were collected for assessment of eNOS and VEGFa expression, cell proliferation, and apoptosis. HUVECs co‐cultured with ADSCs or miR‐ADSCs exhibited higher eNOS and VEGFa protein expression levels compared to DM groups. MiR‐ADSCs enhanced HUVEC proliferation compared to the ADSCs and NC‐miR‐ADSCs. Lower apoptotic rates were observed when HUVECs were co‐cultured with miR‐ADSCs, compared to ADSCs and NC‐miR‐ADSCs. Fifteen male Sprague‐Dawley (SD) rats aged 12 weeks were induced to develop diabetes mellitus by intraperitoneal injection with STZ, and five healthy SD rats were used as normal controls. Eight weeks after developing diabetes, the rats received ADSCs and miR‐ADSCs via injection into the corpora cavernosa, whereas normal controls and DM controls were injected with saline. Erectile function and histological assessment of penile tissues were performed 8 weeks after injection. The ICP/MAP indicated that erectile function was impaired in the DM rats compared with the normal group. Injection of ADSCs and miR‐ADSCs improved erectile function significantly and was associated with the overexpression of eNOS and VEGFa. MiR‐423‐5p knockdown in ADSCs ameliorated high glucose‐mediated damage to HUVECs and improved erectile function in DM rats by inducing eNOS and VEGFa overexpression, indicating that miR‐423‐5p may be a potential target in the treatment of DMED.


| BACKG ROU N D
The morbidity and mortality of diabetes mellitus (DM), which is associated with chronic complications, are continuously increasing. 1,2 Erectile dysfunction (ED) is more likely to happen in men with DM. 3 In different populations and ages, the morbidity rate of ED among men with DM varies from 35% to 90%. 4,5 Diabetes mellitus-induced erectile dysfunction (DMED), which has pathogenic features that include endothelial, neuropathic and microvascular damage and fibrous-muscular alterations, is usually more severe and difficult to treat than nondiabetics, in disregard of the heavy burden. 6,7 Recently, several experimental approaches for DMED have been developed, including insulin treatment, 8 antioxidant therapy, 9 low energy shockwave therapy, 10 stem cells and gene therapy. 11,12 Among these strategies, stem cell-based therapy is considered promising due to its ability to recover functional cells and tissues. In choosing the candidate stem cells from many kinds of mesenchymal stem cells, adipose-derived stem cells (ADSCs) appear to be one of the most suitable types. 3,13 ADSCs have several biological benefits, including a large amount of autologous sources, ease in isolation and the ability to expand. 14 The results of studies in rats with diabetes and cavernous injury, which were treated with the intracavernous injection of ADSCs, showed that erectile function had been restored. [15][16][17] Endothelial nitric oxide synthase (eNOS) plays a key role in penile erection. The blood flow-induced phosphatidylinositol 3-kinase/Akt/eNOS phosphorylation cascade reducing the calcium dependence and sustaining endothelial nitric oxide (NO) release and making the cavernous smooth muscle relax continuously, helps the penile erection maintenance. 18 Endothelial and smooth muscle cells can secrete vascular endothelial growth factor (VEGF) which is a multifunctional glycoprotein. Receptormediated endothelial proliferation can be induced by VEGF in vitro and in vivo. And VEGF is related to endothelial function and an effective vasculogenic and vascular permeability factor. 19 Recently, studies have shown that endogenous NO production can be stimulated by VEGF, which can play positive roles on endothelial and smooth muscle cells, resulting in improvement in erectile function. 20,21 MicroRNAs (miRs) are short non-coding RNA that are 18-23 nucleotides in length. 22 miRs function in regulating gene expression by binding to the 3' untranslated region of the target mRNA. 23 In this study, bioinformatics analysis indicated that miR-423-5p has the ability to regulate both eNOS and VEGFa genes.
Thus, we investigated whether miR-423-5p can improve endothelial cell function and ameliorate erectile function in DM rats, as both eNOS and VEGFa genes play key functions in the mechanism of penis erection. To the best of our knowledge, this is the first study to investigate miRs regulating two important genes in a DMED study.

| Cell culture
Adipose-derived stem cells (ADSCs) and HUVECs were purchased from Procell Life. ADSCs were maintained in stem cell culture medium supplemented with 10% fetal bovine serum and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin). HUVECs were maintained in endothelial cell basal medium supplemented with 1% endothelial cell growth supplement and 10% fetal bovine serum. The glucose concentration of the high glucose medium was 30 mmol/L.

| Animals
Experiments were approved by the institute of Ethics Committee of the third Xiangya Hospital of Central South University. Forty male Sprague-Dawley (SD) rats were acquired from animal center of Central South University. Rats in DM groups were intraperitoneally injected with STZ (60 mg kg −1 ; Sigma-Aldrich) after fasting for 16 h. Fasting blood glucose levels were measured at 3 days after STZ injection using a blood glucose meter. Fasting glucose concentration higher than 16 mmol/L was considered as a success of DM. At 8 weeks after STZ injection, apomorphine (100 μg kg −1 ; Sigma-Aldrich) was used to screen the diabetic rats. Then the DMED rats were randomly divided into three groups (five in each group): DMED control, ADSCs + DMED, miR-423-5p-ADSCs + DMED. And five normal rats with an IC injection of PBS were set as a normal control group. Under aseptic conditions, the determined DMED and normal rats were anaesthetized with 3% diethyl ether. The penis was exposed in each group, and a 24-gauge needle was used to inject a total of 1 × 106 ADSCs or miR-423-5p-ADSCs in 100 μl PBS or only 100 μl PBS into the corpus cavernosum.

| Western blotting
Western blotting (WB) was performed as described previously.
Briefly, the cells and rat penises were lysed in lysis buffer containing protease inhibitors. Protein concentrations of the lysates were determined by the bicinchoninic acid assay (Beyotime Biotechnology). Immunoreactivity was detected after incubation with a horseradish peroxidase-conjugated secondary antibody according to the manufacturer's instructions (Thermo Scientific). GAPDH was used as a loading control. The positive bands were analysed using Gel-pro analyzer software, and integrated optical density (IOD) was measured.

| ELISA
The cell culture medium was collected after coculture, the supernatant was collected by centrifugation for 10 min at 1500 rpm. eNOS and VEGFa expression levels were measured using an ELISA kit (Solarbio Life Sciences) according to the manufacturer's instructions (SEA868Ra for eNOS, SEA143Ra for VEGFa). Absorption at a wavelength of 450 nm (A450) was determined using the microplate reader.

| Masson trichrome stain
Masson trichrome staining using a Trichrome Stain (Masson) Kit (Sigma-Aldrich Co.) was performed to visualize fibers in tissues, following the manufacturer's instructions. Briefly, the tissue slides were deparaffinized, stained in preheated Bouin's solution, and washed in running tap water to remove the yellow colour from sections. Then, the slides were respectively stained in Working Weigert's Iron Hematoxylin Solution, Biebrich Scarlet-Acid Fucshin,

Working Phosphotungstic/Phosphomolybdic Acid Solution and
Aniline Blue Solution. The stained slides were observed under an optical microscope (magnification 40×).

| Intracavernosal pressure (ICP) measurement
Erectile function was determined by intracavernosal pressure (ICP) and mean arterial pressure (MAP) 4 weeks post-injection. Under 3% pentobarbital sodium, the major pelvic ganglion (MPG) and cavernous nerves (CN) were exposed by midline laparotomy. The penile was exposed by removing overlying skin and ischiocavernosus muscle. One of the 24-gauge needles that were connected to PE-50 tubes with heparinized saline (250 IU ml −1 ) was inserted into the left carotid to measure MAP. The other one was inserted into corpus cavernosum (CC) to measure ICP. PE-50 tubes were connected to the data acquisition system (MP150, BIOPAC Systems Inc.). The CNs were stimulated using a stainless steel bipolar hook electrode with the following parameters: 20 Hz, pulse width of 0.2 ms, 1.5 mA, for 50 s. The ratio of maximal ICP (mm Hg) to MAP (mm Hg) was calculated.

| Statistical analysis
All statistical analyses were performed with SPSS 19.0 (SPSS Inc.).
All results are expressed as the mean ± standard deviation (SD).
Multiple comparisons between groups were performed using ANOVA followed by post hoc analysis using the Tukey-Kramer test, whereas a comparison between two groups was performed using a t-test. Differences with a p < 0.05 were considered statistically significant.

| MiR-423-5p directly targets eNOS and VEGFa mRNAs
Bioinformatics prediction revealed that miR-423-5p has one specific potential binding site for eNOS and VEGFa mRNAs within the 3'-UTR ( Figure 1A). There are seven base-pairs at the binding site for eNOS mRNA and miR-423-5p, and eight for VEGFa mRNA and miR-423-5P. A luciferase assay was performed to validate this prediction. Both eNOS and VEGFa luciferase activity were suppressed relative to the control; the suppression of eNOS was 66%, whereas VEGFa was 60% ( Figure 1B). These findings indicate that eNOS and VEGFa are target genes of miR-423-5p.

| Knocking out miR-423-5p in ADSCs alleviates high glucose-induced damage in HUVECs
We transfected ADSCs with the miR-423-5p inhibition lentivirus, and then miR-ADSCs were co-cultured with HUVECs. In normal glucose conditions, ADSCs, NC-miR-ADSCs and miR-ADSCs promoted eNOS and VEGFa expression in HUVEC conditioned media ( Figure 3A,B). miR-ADSCs increased the protein expression of eNOS and VEGFa under high glucose conditions compared to ADSCs and NC-miR-ADSCs ( Figure 3A,B). A CCK-8 assay was performed to assess cell proliferation. miR-ADSCs promoted HUVEC proliferation under both normal and high glucose conditions ( Figure 3C). eNOS and VEGFa are key proteins in penis erection. Therefore, western blotting was performed to evaluate eNOS and VEGFa expression. miR-423-5p inhibition in ADSCs was associated with increased HUVEC eNOS and VEGFa expression. This increase was more profound under high glucose concentrations than under normal glucose concentrations ( Figure 3D-F). To further investigate whether the inhibition of miR-423-5p in ADSCs would benefit HUVECs, cell flow cytometry was also conducted. Propidium iodide (PI) was used to assess cell apoptosis. The number of late apoptotic cells caused by high glucose damage sharply decreased when cocultured with miR-ADSCs ( Figure 3G). Reduced VEGFa signaling and impaired angiogenesis as well as collateral blood vessel formation occur in patients with diabetes mellitus. 29 Several groups have shown that humans and rats with ED have lower VEGFa expression than normal humans and rats, suggesting VEGFa could be a target for the treatment of ED. VEGFa also enhances the proliferation of cavernous smooth muscle cells and endothelial cells, further supporting erectile function. 30 VEGFa can be induced by a NO synthesis pathway that also facilitates angiogenesis. 31 Impaired vasculogenesis was reported in eNOS knockout mice (eNOS −/− ). 32 Diminished wound healing was also detected due to reduced VEGF-mediated migration. 33 ADSCs are somatic stem cells with multipotency and little immunogenicity. 34 Moreover, ADSCs have been used to treat many diseases including the repair of muscular tissue. 35 Several groups have investigated the feasibility and advantage of using ADSCs for ED therapy in rat models. 36,37 Implantation of ADSCs has been shown to significantly improve erection function in diabetic ED rats. 12 MicroRNAs are small, non-coding RNAs that modulate gene expression by binding to the 3'UTR of the target gene. 38 MicroRNAs affect many biological processes, including cell apoptosis, proliferation and metabolism. 39 A microRNA may not have a single target gene, instead influencing multiple genes simultaneously. We hereby describe an approach for improving eNOS and VEGFa expression for the potential treatment of DMED using microRNAs. MiR-423-5p can promote gluconeogenesis and hyperglycaemia. 40 A computational biology study showed that miR-423-5p was at a high express level in the obesity and type 2 diabetes adipose tissue. 41 Both hyperglycaemia and obesity are bad effects on erectile function.

| Erectile function assessment
In this study, we found that miR-423-5p simultaneously affected the expression of both eNOS and VEGFa. Knockout of miR-423-5p expression in ADSCs was associated with an increase in eNOS and VEGFa expression. ADSCs may differentiate into local smooth muscle cells or endothelial cells to restore organ function. ADSCs can also exert a local effect by secreting cytokines and growth factors. 17,36 In this study, ELISA and western blot results demonstrated that HUVEC co-cultured with miR-ADSCs showed overexpression of eNOS and VEGFa. As these two proteins play a key role in erection, we injected miR-ADSCs and ADSCs into diabetes-induced ED rats to evaluate their effect on erection. Both miR-ADSC-and ADSC-treated diabetic rats had improvement in erectile function. The miR-ADSC-treated group showed greater improvement than the ADSC-treated group.
Together, these findings indicate that knocking out miR-423-5p relieves the inhibition of eNOS and VEGFa expression in ADSCs, thereby supporting penile erection.
Our results were similar to previous studies in that we also used stem cells in treating DM rats. However, the present study has limitations, and our findings require further validation. We did not assess fibrosis factors that may cause tunica albuginea diseases such as Peyronie's disease. In addition, the mechanism by which ADSCs affect HUVECs warrants further investigation, including whether ADSCs secrete exosomes.

| CON CLUS IONS
Knocking down miR-423-5p in ADSCs ameliorated the high glucose damage in HUVECs and improved erectile function in DM rats.
There was an associated overexpression of eNOS and VEGFa, suggesting that miR-423-5p may be potentially used as a target in cell therapy for DM associated erectile dysfunction.

ACK N OWLED G EM ENTS
This work was supported by the National Natural Science Foundation of China (No. 81571432 and No. 82071636).

CO N FLI C T O F I NTE R E S T
All authors declare no competing financial interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
All datasets used during the current study are available from the corresponding author on reasonable request.