Mesenchymal stem cell‐derived exosomes ameliorate erection by reducing oxidative stress damage of corpus cavernosum in a rat model of artery injury

Abstract Erectile dysfunction (ED) is a common ageing male's disease, and vascular ED accounts for the largest proportion of all types of ED. One of the mechanisms of vascular ED in the clinic is arterial insufficiency, which mainly caused by atherosclerosis, trauma and surgical. Moreover, oxidative stress damage after tissue ischemia usually aggravated the progress of ED. As a new way of acellular therapy, mesenchymal stem cell‐derived exosomes (MSC‐Exos) have great potential in ED treatment. In the current study, we have explored the mechanism of MSC‐Exos therapy in a rat model of internal iliac artery injury‐induced ED. Compared with intracavernous (IC) injection of phosphate‐buffered saline after artery injury, of note, we observed that both mesenchymal stem cells (MSCs) and MSC‐Exos through IC injection could improve the erectile function to varying degrees. More specifically, IC injection MSC‐Exos could promote cavernous sinus endothelial formation, reduce the organization oxidative stress damage, and improve the nitric oxide synthase and smooth muscle content in the corpus cavernosum. With similar potency compared with the stem cell therapy and other unique advantages, IC injection of MSC‐ Exos could be an effective treatment to ameliorate erectile function in a rat model of arterial injury.

old. 3 Common mechanism of vascular ED in the clinic is arterial insufficiency, which mainly caused by atherosclerosis, trauma and surgical. Many studies have proved that in arterial ED, as the corpus cavernosum tissue continues to be stimulated by the ischemic and hypoxic environment, the release of reactive oxygen species in the penis increases. When the harmful compounds produce more than the anti-oxidative defence ability of the tissue, free radical accumulation may cause cell degeneration, cavernosum vascular endothelium and nerve damage, thereby further increasing the progress of ED. [4][5][6] Previous studies have proved that antioxidant therapy is of great significance for arterial ED. 7 Phosphodiesterase type 5 inhibitors (PDE5is) are the first-line treatment for ED. However, the efficiency of PDE5is must base on the integrity of corpus cavernosum vascular endothelial function and bioavailability of the nitric oxide (NO). 8 Given these reasons, it is difficult to achieve satisfactory results for those patients with more serious refractory ED. On the other hand, PDE5is are expensive and have serious adverse effects. 9 Thus, it is necessary for exploring other strategies with better efficacy for treating ED, especially for arterial ED.
Bone mesenchymal stem cells (MSCs) are pluripotent cells with strong differentiation ability under different conditions. Stem cells were originally considered as substitutes for defective or damaged cells in the past. More recently, however, various types of stem cells have proven to influence the host environment due to they can secrete various bioactive molecules by the paracrine route. 10,11 Stem cells could secrete a large number of biological factors, which have nutrition, angiogenetic, antifibrotic and inflammatory modulating properties. Moreover, as a method of stimulating the activity of tissue resident receptor cells, stem cells are also able to secrete nucleic acids, lipids and proteins in the extracellular microvesicles. [12][13][14] The exosomes mainly refer to particles between 40 and 150 nm in diameter among the many types of extracellular microvesicles. 15 According to the latest research, exosomes derived from stem cell are efficacious in animal models of ED, which contains acute cavernous nerve injury 16 and type 2 diabetes. 17 However, whether transplant MSC-derived exosomes (MSC-Exos) can be exploited to recover arterial injury ED, and the mechanisms remain largely undetermined. As tissue will continue to be exposed to risk factors during vascular events, compare with the protective effect of exosomes on anti-apoptosis after tissue injury, 16,17 we found that this therapeutic effect might mainly delay the damage of cavernosum by promoting tissue repair. In the current study, we have explored the treatment efficacy of intracavernous (IC) injection of MSCs or MSC-Exos in a rat model of arterial injury ED and further elucidate the underlying mechanism of MSC-Exos treatment. All the rats were randomly divided into five groups of six rats each. The sham group had low abdominal incision but without internal iliac artery ligation. The remaining animals were subjected to bilateral internal iliac arteries ligation to establish arteriogenic ED models. 18 Four weeks later, the 24 rats were randomly divided into four groups. The PBS group received IC injection of phosphatebuffered saline (PBS); the MSCs and MSC-Exos groups received IC F I G U R E 1 Establishment the rat model of artery injury erectile dysfunction. A, Expose and dissociate the internal iliac artery. B, Bilateral internal iliac arterial ligation

A B
injection of MSC or MSC-Exo separately. At the fourth week after IC injection, animals' erectile function was measured and the penile tissues were harvested for histologic analysis and western blotting.

| Bilateral internal iliac arterial ligation surgery
The ligation surgery was performed under anaesthesia with 45 mg/ kg pentobarbital sodium, body temperature of each animal remains stable. A 4-5 cm transverse incision in lower abdomen was made, then under a dissecting microscope, deep below the superior vesical artery surface free internal iliac artery ( Figure 1A). After bilateral internal iliac arteries were ligated ( Figure 1B), the organ reduction and incision was sutured. Intramuscular injection of 210 mg/kg/d cefazolin sodium pentahydrate to prevent infection in the first 3 days after surgery.

| Erectile function evaluation
Erectile function was assessed based on changes in intracavernous pressure (ICP) after the cavernous nerves electrostimulation. 19 Under anaesthesia with intraperitoneal injection pentobarbital sodium (45 mg/kg), ICP was measured with a 25-G heparin-filled needle, which inserted into the corpora cavernosum of the penis. The major pelvic ganglia were identified through a lower abdominal midline incision and then the cavernous nerves were exposed, the animal nerve-stimulating electrode (3-mm apart, ADINSTRUMENTS, MLA0320) was connected to the cavernous nerve. The stimulus parameters were as follows: 10-V amplitude, 15-Hz frequency, 5ms pulse width and 60-seconds duration. The electrical stimulation was done in triplet, with a 5-minutes interval between the subsequent stimulation to ensure stability maintains for each animal. 20 In the meantime, the right internal carotid artery was detached and inserted into a 24-G vein indwelling needle to measure mean arterial pressure (MAP). All blood pressure change data were recorded by the ADInstruments PowerLab 16/35 workstation and analysed by LabChart 8 Software (ADInstruments). The ratio of maximal ICP to MAP was calculated to normalize for variations in systemic blood pressure.

| Histologic analysis
After erectile function was evaluated, the penile midshaft at the injection site was harvested. One-third of the tissues were fixed in 4% paraformaldehyde, then dehydrated and embedded in paraffin, and the rest fresh tissue was frozen in liquid nitrogen for other assays.
The tissues were coronally cut to 5-µm thickness for immunofluo-

| Western blotting
Western blot (WB) was performed as previously described. 21 Primary Densitometric analysis of the bands was implemented using ImageJ software (National Institutes of Health).

| Statistical analyses
The results were analysed using GraphPad Prism software (version 7.0, GraphPad Software) and expressed as mean ± standard error of the mean. Multiple groups were compared using oneway analysis of variance followed by the Student-Newman-Keuls test for post hoc comparisons. Statistical significance was set at P < .05.

| Characterization of MSCs and MSC-exos
The morphology of MSCs was observed under a light microscope, which manifests fusiform, homogenous fibroblastic morphology ( Figure 2A). Transmission electron microscopy showed that MSC-Exos were cup-shaped double-layer membrane structure with a diameter in the range of 50-200 nm ( Figure 2B). Nanoparticle Tracking Analysis demonstrated that the average diameter of the particle was about 115 nm; the main peak of particle size was 74 nm; and the concentration of the particle was 1.11 × 10 10 particles/mL ( Figure 2C). WB analysis of MSC-Exos showed the existence of exosome characteristic marker proteins TSG101 and CD9 ( Figure 2D).

| Effects of MSC-Exos on NO/cGMP signalling pathway
In corpus cavernosum tissues, nNOS and eNOS are the dominant drivers of penile erection, which could catalyse the NO released during non-cholinergic, non-adrenergic neurotransmission and from the endothelium, thereby activating cyclic guanosine monophosphate (cGMP) signalling pathway. 22 As the WB result show ( Figure 5A), the levels of nNOS and eNOS in the treated groups were higher than those in the PBS group (P < .05 for all), but lower than the sham group (P < .01 for all; Figure 5B,C). The iNOS was also an important factor in erection. It was reported that the production of iNOS is closely related to the existence of inflammatory environment in the cavernosum. 23 The data revealed that iNOS expression was higher in the PBS and MSC-Exos groups than in the sham group (P < .01), but treat with MSC-Exos could decrease the expression of iNOS in comparison with the PBS group (P < .01; Figure 5D).  Figure 5G).

| MSC-Exos treatment improves the ratio of smooth muscle to collagen and smooth muscle content in the corpus cavernosum
According to the statistical result of Masson's trichrome staining ( Figure 6A), the ratio of cavernosum smooth muscle to collagen was 0.081 ± 0.008, 0.066 ± 0.007, 0.048 ± 0.008, 0.057 ± 0.009 in the sham group, MSCs group, MSC-Exos (low) group and MSC-Exos (high) group, respectively, which all higher than the PBS group (0.026 ± 0.07, P < .01 for all; Figure 6F). Corpus cavernosum smooth muscle content was detected by immunohistochemical staining and Western blot ( Figure 6B,C). The penile tissue from the PBS group showed significantly lower α-SMA-positive staining area compared with the sham group (P < .01). Both MSCs and MSC-Exos treat exhibited partial but significant restoration of smooth muscle content after bilateral internal iliac arterial ligation, and similar results were observed by WB analysis (Figure 6E,D).

F I G U R E 4 IC-injected MSCs or MSC-Exos promote cavernous endogenous stem cells differentiate into cavernous sinus endothelial cells.
A, Representative images of western blots for CD31 and VEGFA in cavernosum from each group. B and C, Data are presented as the relative density of CD31 and VEGFA compared with that of GAPDH. Each bar depicts the means ± standard deviation from n = 6 animals per group. *P < .05 vs the PBS group, **P < .01 vs the PBS group, #P < .05 vs the Sham group, ##P < .01 vs the Sham group. D, Representative immunofluorescence staining of CD31 (red) and OCT4 (green) in a penile midshaft specimen from each group. The dotted line represents the contour of the cavernous sinus. Original magnification, ×200

| The perimeter changes of cavernosum
Bilateral internal iliac arterial ligation caused corpus cavernosum atrophy (Figure 7), compared with the sham group, the cavernosum perimeter of the PBS group was statistically de-

| D ISCUSS I ON
In the current study, we found that MSC-Exos could effectively improve the erectile function in a rat model of arterial injury. More specifically, IC injection exosomes could ameliorate erection by promoting cavernous sinus endothelial formation and reduce the organization oxidative stress damage. Although MSCs are highly capable of differentiation, the paracrine mechanism of MSCs is currently considered to play a major role in therapy. 11 Exosome of MSC is an important bioactive substance vector which contains multiple types of nucleic acids, lipids and proteins. 12 They have been demonstrated to involved in multiple pathologic and physiologic processes, which include proliferation and differentiation, 24 anti-fibrosis, 25 immunomodulation 26 and tissue regeneration. 27 In our study, exosomes were isolated from the culture supernatants of MSCs, and these pellets proved could ameliorate rat's erectile function independently with similar potency compared with the MSCs group.
Erectile dysfunction, a multifactorial condition with a complex neurovascular process, can be observed typically in patients with endocrinologic, neurogenic, vasculogenic, cavernosal diseases and drug-induced. 28 In order to simulate clinically common vascular ED, we established an arterial injury ED model by ligating the bilateral internal iliac artery of rats. 18,29 Despite the formation of vascular collateral circulation after ligation, the blood supply to the corpus cavernosum cannot be completely blocked, but the reduction of blood supply will cause the cavernosum tissue continues to be stimulated by the ischemic and hypoxic environment, F I G U R E 6 Treatment improves the ratio of smooth muscle to collagen and smooth muscle content in the corpus cavernosum. A, The smooth muscle (red) and collagen (blue) tissues stained by Masson's trichrome staining. Original magnification, ×20 and × 100. B, Immunohistochemical expression of α-SMA in corpus cavernosum. Original magnification, ×100. C, Representative images of western blots for α-SMA in cavernosum in each group. D, Western blots' data are presented as the relative density of α-SMA compared with that of GAPDH. E, Immunohistochemically semi-quantitative data of the proportion of α-SMA positive expression area. F, The semi-quantitative data of the ratio of smooth muscle area to collagen area. Each bar depicts the means ± standard deviation from n = 6 animals per group. *P < .05 vs the PBS group, **P < .01 vs the PBS group, #P < .05 vs the Sham group, ##P < .01 vs the Sham group thereby increasing the release of reactive oxygen species in the penis. Many studies have suggested that oxidative stress is the main factor of ED progress during penile ischemia, 6,7 which may lead to cell apoptosis, cavernosum vascular endothelium and nerve damage. 30 It has been reported in the literature that exosomes released by stem cells can improve erectile function in a rat model of cavernous nerve injury and type 2 diabetes by anti-apoptosis. 16,17 According to the histological analysis of penis 1 month after IC injection, we found that the possible mechanism of MSC-Exo alleviating oxidative stress damage might be through the mobilization of cavernous endogenous stem cells to differentiate into endothelial cells. MSCs have been shown to promote tissue angiogenesis by paracrine the vascular endothelial growth factor (VEGF) or exosome containing VEGF and specific miRNA. 31,32 Vascular endothelial growth factor is a highly specific factor that has been shown to stimulate endothelial cells migrations and differentiation. It is reported that VEGF alone or combined with other factors can induce stem cells to undergo to endothelial cell differentiation. These cytokines have fully proved to have the ability to induce stem cell differentiation in vitro. 33 As a marker of stem cell, the Octamer-binding transcription factor 4 (OCT4) not only expressed in embryonic stem cells, but also reported to widely exist in adult stem cells in recent years. It is related to the pluripotency maintaining and regulating the differentiation ability of stem cells. 34 43,44 However, due to the inclusions of exosome are very complex, there are also related researches that attribute its active ingredients to miRNAs. As a key factor in exosomes facilitating VEGF signalling, miRNA-126 in MSC-Exo has been proved to have a great effect on promoting angiogenesis. 45,46 All of this suggests that different inclusions in exosomes may synergistically enhance the therapeutic effect, the specific mechanisms of the MSC-Exo involved in the repair of injury remain need to be further identified.

| CON CLUS IONS
Our results provide the evidence that intracavernous injection MSCderived exosomes could ameliorate erectile function by reducing the oxidative stress damage of corpus cavernosum in an arterial injuryinduced ED rat model, which further has great potential in the clinical treatment of severe arterial injury ED.

ACK N OWLED G EM ENTS
This work was supported by the grants from Science and Technology Planning Project of Guangdong Province (No.2017B030314108).

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.