Erectile dysfunction and exosome therapy

Erectile dysfunction (ED), as a common male disease, can seriously reduce the life quality of men and their partners. With the improvement of human living standards, ED is considered to be an important health issue that plagues men. However, it is difficult for existing therapeutic approaches to meet the needs of all patients, so it is necessary to develop novel treatment strategies. Exosomes, as a class of vesicles secreted by cells with bilayer membrane structure, are involved in various physiological and pathological processes in human body and considered to have great therapeutic potentials. This review summarizes the recent advances on exosome therapy with animal models of ED, and proposes the prospect of future research in order to provide a basis for clinical trials and clinical translation.

35% of patients do not respond to the pharmacological therapy (1). As current treatments do not provide maximum benefits to patients, it is crucial to investigate novel strategies for ED treatments (9). Extracellular vesicles (EVs) are cell-derived membrane structure with a diameter of 40 nm to 1000 nm. Exosomes and microvesicles are two types of EVs that are released from the endosomal system or shed from plasma membrane, respectively (10). Exosomes are nanosized particles with a diameter of roughly 40-160 nm secreted by various cells under physiological or pathological conditions (11). Exosomes have a bilayer structure that is made by the plasma membrane through encapsulating extracellular components and membrane proteins as well as intersecting with other vesicles and organelles (10, 12). Therefore, exosomes contain many constituents such as metabolites, proteins, lipids and nucleic acids (13,14). Exosomes are particularly heterogeneous population due to their origin from different cells, as well as the notable differences in their size, composition and effect on the function of recipient cells (15,16). Exosomes are involved in a variety of physiological and pathological processes in the human body (17), and can be exploited as molecular and signal carriers in intercellular communication (18). Exosome biogenesis enables cells to rapidly and selectively remove proteins from the plasma membrane, facilitating procedures like sperm-egg binding (19). Exosomes are linked to the process of viral infection and are involved in the induction of innate and adaptive immune responses (20). Studies on exosomes and diseases have revealed that exosomes may be involved in cardiovascular and metabolic disorders, take a role in the pathogenesis of neurological disorders, and dynamically affect the growth of cancers (11,21).
Exosomes are frequently investigated in clinical studies. Exosomal non-coding RNAs (ncRNAs) have been revealed to be expressed differently in most human disorders, which paves the way for their potential application as biomarkers in early stage of diseases (22). Exosomes also serve as drug delivery systems (DDS) for the treatment of cancer (23). For instance, exosomes containing cisplatin inhibited the progression of hepatocellular carcinoma (24). In animal experiments, transplanted bone marrow promoted the regeneration of injured b cell by releasing exosomes enriched with miR-106b-5p and miR-222-3p (25). Exosomal miRNAs produced by mesenchymal stem cells have been found to exhibit antiatherosclerotic properties (26). Therefore, it is valuable to further explore the application of exosomes in ED patients, which might be caused by diabetes or cardiovascular disease.
In this review, we introduced the biosynthesis and contents of exosomes, and concentrated on the application of exosomes as well as other types of EVs in the treatment of ED.

The biogenesis of exosomes
The exact mechanisms of exosome formation are still unclear, but existing studies suggest that the process of exosome biogenesis may be similar in different cell types. Generally, the biogenesis of exosomes is a continuous dynamic process and consists mainly of two invaginations of plasma membrane and the formation of multivesicular bodies (MVBs) containing intraluminal vesicles (ILVs) (11).
First of all, exosomes originate from the inward budding of plasma membrane (that is the first invagination of plasma membrane) to form early sorting endosomes (ESEs) with cell surface proteins and extracellular constituents (27,28). In some cases, the generated ESEs may be directly merged with the preformed ESEs (11). Afterwards, ESEs either fuse with the plasma membrane for the recycling of sequestered cargoes or convert into late sorting endosomes (LSEs), which can give rise to MVBs with the involvement of endoplasmic reticulum and Golgi complex (29,30). During the maturation of MVBs, cytoplasmic cargoes including nucleic acids, proteins, lipids, amino acids and metabolites can enter into LSEs by inward budding of endosomal membrane (that is the second invagination of plasma membrane), which leads to the generation of ILVs (27,(29)(30)(31). Finally, some MVBs are degraded by the autophagosome or lysosome to maintain cellular homeostasis, while others fuse with the plasma membrane via the exocytotic pattern to secrete ILVs, which are also considered as exosomes (32,33). Notably, exosomes derived from other cells can be taken up by the cells and further fused with ESEs (11).
However, in addition to this canonical model, several studies also revealed the other mechanisms involving in the biogenesis of exosomes. Exosomes can be formed immediately by outward budding through the plasma membrane or delayed released by budding through the deep invagination of plasma membrane (17). Electron microscopy experiments in mesenchymal stem cells and human immune cells have confirmed the existence of the above phenomenon (17,(34)(35)(36).
After exosomes are released by donor cells, they either fuse directly with the plasma membrane of recipient cells to transfer cargoes, or interact with the receptors of recipient cells to activate the corresponding signaling pathways, or undergo endocytosis by recipient cells to release cargoes or fuse with the endosomes to take part in the biogenesis of exosomes in recipient cells (11,27,31).

The contents of exosomes
Exosomes contain not only extracellular substances and plasma membrane proteins, but also a variety of cytoplasmic substances such as DNAs, RNAs, proteins, lipids and metabolites, some of which can be used as biomarkers of exosome (27,30,31). The content of exosomes is not constant and varies widely in different cell microenvironments.
Exosomal nucleic acids include DNA, mRNA and non-coding RNA (miRNA, lncRNA and circRNA), among which miRNA is one of the most abundant nucleic acids in exosomes. More and more miRNAs contained in exosomes are identified in scientific researches, and they regulate the function of recipient cells or the expression of target mRNA in recipient cells (22,32). The overexpression of miR-934 in colorectal cancer cell-derived exosomes induced M2 macrophage polarization, activated PI3K/ AKT signaling pathway and decreased the PTEN expression (48). High levels of miR-205 were found in circulating exosomes from ovarian cancer patients promoting the angiogenesis and tumor growth (49). Moreover, it was reported that exosomal miRNA from M2 macrophages such as miR-328 promoted the proliferation of pulmonary interstitial fibroblasts and induced the development of lung fibrosis (50). In addition, lipids including cholesterol, ceramides, sphingomyelin and phosphatidylinositol are also contained in exosomes (27).

The therapeutic potential of exosomes in ED
Most studies on exosomes have focused on the field of oncology, while the correlation between exosomes and ED is less studied. The investigation of effects of exosome on the amelioration of ED was analyzed for the first time in 2017 (51). Details of exosome therapy studies in ED models are displayed in Figure 1 and Table 1. Schematic diagram of the molecular mechanism of exosome therapy for ED. CCSMC, corpora cavernosum smooth muscle cell; eNOS: endothelial nitric oxide synthase; nNOS: neuronal nitric oxide synthase; iNOS: inducible nitric oxide synthase; Ang 1, angiotensin 1; Ang 2, angiotensin 2; ANP: atrial natriuretic peptide; BNP: brain natriuretic peptide; TGF-b1, transforming growth factor-b 1; NT3: neurotrophin 3; NGF, nerve growth factor.

Diabetic ED
ED is considered as one of the long-term complications of diabetes, which increases the risk of developing ED by 2.5-fold, and more than 50% of people with diabetes are affected by ED (68). Diabetic patients are mainly manifested with hyperglycemia and insulin resistance, usually accompanied by the metabolic syndrome (obesity, hypertension, dyslipidemia), hypogonadism, cardiovascular diseases and neuropathy (69). These manifestations and comorbidities affect the levels of androgen, inflow of arteries, outflow of veins as well as nerve signaling, and ultimately influence the production of reactive oxygen species, NO, cyclic guanosine monophosphate (cGMP) and nitric oxide synthase (NOS), and the function of endothelial cells and corpus cavernosum smooth muscle cells (CCSMCs) during the erection of penis (69,70).
Chen et al. established a rat model of type II diabetes and isolated exosomes from adipose-derived stem cells (ADSCs) by ultracentrifugation (51). They found that ADSC-derived exosomes promoted the recovery of erectile function by increasing the ratio of maximal intracavernous pressure (ICP) to mean arterial pressure (MAP), elevating the endothelium and smooth muscle contents and decreasing the apoptosis in cavernous endothelial cells and smooth muscle cells (51). In addition to type 2 diabetes, ED was also induced following type 1 diabetes. Zhu et al. injected type 1 diabetic ED rats with three different doses of ADSC-derived exosomes (0, 10 or 100 mg, respectively) (52). It was demonstrated that exosomes improved the erectile function by reversing fibrosis, increasing endothelial content and angiogenic activity in a dose-dependent manner. Interestingly, they also performed miRNA sequencing on the extracted exosomes and found that some functional miRNAs were contained in these exosomes, including proangiogenic miRNAs (miR-126, miR-130a, miRNA-132) and antifibrotic miRNAs (miR-let7b and miR-let7c). Although the role of these miRNAs in exosomes in the treatment of ED has not been validated in vitro or vivo, these findings revealed the potential mechanisms of exosome therapy in ED, which opened up a new perspective for the future investigation of exosomes in the treatment of ED.
Notably, donor cells can be modified by transgenic methods so that exosomes released by these cells can contain a large number of specific cargoes. For example, bone marrow-derived stem cells (BMSCs) treated with miR-21-5p-agomir resulted in the increase of miR-21-5p in BMSC-derived exosomes, which decreased the expression of target gene programmed cell death 4 (PDCD4) in CCSMCs, attenuating the erectile dysfunction through leading to the proliferation and apoptosis inhibition of these cells (53). Meanwhile, exosomes derived from ADSCs transfected with corin siRNA promoted the neurovascular function and suppressed the levels of inflammatory factors including TNF-a, IL-1b and IL-6 (54). Our group also investigated the therapeutic effects of exosomes in ED. Distinct from the above study, we isolated exosomes from CCSMCs and found that this type of exosomes was more easily retained in the corpus cavernosum and better ameliorated the diabetes-induced erectile dysfunction compared with exosomes from ADSCs and BMSCs (55).

Bilateral cavernous nerve injury-induced ED
Prostate cancer surgery and other pelvic surgeries often result in damages to the cavernous nerve, which originates from the major pelvic ganglion (MPG) and controls the relaxation and contraction of CCSMCs, thus regulating the erection of the penis (71). It was reported that up to 80% of prostate cancer patients suffer from ED after radical prostatectomy (72). Many molecules and signaling pathways contribute to the development of ED during the injury of cavernous nerve, for instance TGF-b, hydrogen sulfide, NO pathway, RhoA/ROCK pathway or oxidative stress-related pathway (73, 74). The dysregulation of these factors after BCNI leads to the tissue fibrosis, as well as phenotypic transformation and apoptosis of CCSMCs (73).
Exosomes isolated from ADSCs and BMSCs have been applied not only to diabetic ED but also to BCNI-induced ED. Ouyang et al. found that four weeks after injection with exosomes into the corpus cavernosum of BCNI-induced ED rats, the erectile function was obviously improved via inhibiting apoptosis in CCSMCs (59). Similarly, ADSC-derived and BMSC-derived exosomes have been shown to be effective in recovering erectile dysfunction in BCNI rat model (58). Both of them can alleviate the distortion of normal neural anatomy, smooth muscle atrophy and collagen deposition, which impaired the erection of penis.
In order to enhance the uptake of exosomes and the efficacy of treatment, some research teams have developed specific hydrogels and mixed them with exosomes for the treatment of ED. Liang et al. fabricated polydopamine thermosensitive hydrogels, which exhibited sol-gel transition at body temperature and allowed exosomes to be released slowly within two weeks (56). In vivo experiments confirmed that ADSC-derived exosomes loaded within polydopamine thermosensitive hydrogel improved the erectile function by healing the endothelial cells and neurons in penis. On the other side, ADSC-derived exosomes encapsulated into the thermosensitive hydrogel significantly repaired the cavernous nerves injury in rats, thus restoring erectile function (57).
In addition, a study showed that Schwann cell-derived exosomes promoted nerve regeneration of MPG and cavernous nerve with MPG, suggesting that they may provide potential therapeutic options for ED treatment (75). Since this study only involved ex vivo experiments, appropriate in vivo experiments are needed to further confirm the findings.

Other types of ED
So far, exosomes have been introduced to treat some other types of ED in addition to diabetic ED and BCNI-induced ED. Vascular ED accounts for a large proportion in elderly patients with ED, which can be caused by atherosclerosis, trauma and surgery. It has been shown that in internal iliac artery injury-induced ED rats, BMSC-derived exosomes promoted cavernous endogenous stem cells to differentiate into cavernous sinus endothelial cells while effectively reduced the oxidative stress damage of corpus cavernosum (60). These findings provided a novel insight and strategy for the clinical treatment of severe arterial injury ED.
Since erectile dysfunction is often observed in patients with obstructive sleep apnea, the researchers developed a rat model of chronic obstructive hypoxia-induced ED. The miR-301a-3penriched ADSC-derived exosomes affected the apoptosis and autophagy of CCSMCs by targeting PTEN and TLR4, and ultimately improved erectile function (62). Furthermore, Yang et al. demonstrated that human urine-derived stem cell (HUSC)derived exosomes ameliorated the fibrosis in tunica albuginea and restored erectile function in Peyronie's disease rats (61).

The therapeutic potential of other subsets of EVs in ED
Due to the low production of EVs, Kwon et al. developed embryonic stem cell (ESC)-derived EV-mimetic nanovesicles (ESC-NVs), and diabetic mice were received intravenous injection of 0.1, 0.5, 1, 2 or 5 mg ESC-NVs for 2 times, respectively. The results revealed that those ESC-NVs enhanced penile neurovascular regeneration by boosting the expression of angiogenic and neurotrophic factors (63). A team of researchers from South Korea extracted EV-NVs from cavernous pericytes, and they found that these NVs can significantly promote neurovascular regeneration and ameliorate erectile dysfunction in both diabetic and BCNI-induced ED rats (64, 65). The studies also compared the efficacy of different doses of NVs and found that the higher the dose, the better the recovery of pathological changes and erectile dysfunction. Moreover, EVs derived from HUSC (HUSC-EVs) have been proven to ameliorate erectile dysfunction effectively (66,67). In contrast to the conventional intracavernous or intravenous injection, Zhuang et al. mixed HUSC-EVs with hyaluronic acid and smeared them on the glans of the rats several times (67). The results showed that this administration can also improve apoptosis, angiogenesis, and smooth muscle regeneration as the conventional injection Table 2.

Future prospects
Basic and preclinical researches have shown that exosomes have the potential to treat ED. Since the research in this field is still at a preliminary stage, there are no ongoing or completed clinical trials. Although exosomes exhibit good therapeutic effects in rat and mouse, rodent models are insufficient to predict human clinical outcomes. Exosome therapy in ED needs to be validated in higherorder animals that more closely mimic the physiological and clinical characteristics of the human body. In fact, a large number of clinical trials based on exosomes have been completed in many other diseases (76)(77)(78)(79)(80)(81)(82)(83), so we need to speed up the pace to more comprehensively confirm the therapeutic effect of exosomes on ED and promote the translation from animal studies to clinical trials. In addition, it is worth noting that most of the existing studies only focus on the therapeutic effects of exosomes on ED and related mechanisms, but there are relatively few explorations of exosome contents. In the future, we should take actions to further understand the role of key molecules identified in exosomes in the treatments of ED.
There are still several key issues to be resolved, including longterm safety, optimal source of exosomes, optimal therapeutic method, dose and course, appropriate delivery system and elucidation of the specific mechanism. Most of the studies used exosomes derived from allogeneic cells to treat ED through intracavernous injection. Tail vein injection, intratunical injection and nerve injury in situ injection are also working with exosomes during the treatment of ED, although there are only a few reports in the literature. Considering the low yield of human exosomes, how to obtain sufficient exosomes for future clinical use is worth thinking. The sources of exosomes and therapeutic doses need recognized standards, and the comparison of the effects of exosomes from different sources requires more rigorous evidence to support it. Meantime, the doses of exosomes in previous studies vary greatly, which makes the appropriate dose necessary. Most researchers have injected exosomes at a dose of 100 mg and achieved good results, but this only applies to rodent models. Despite the fact that exosomes do not express MHC proteins and will not cause immune tolerance and cell malignancies (84), it is still unknown whether there will be other adverse advents in the further research owing to the fact that the contents of exosomes have not been fully clarified. While exosomes may not be able to completely reverse the pathological changes in the corpus cavernosum, they can delay the progression of the disease, which is enough to improve sexual function and improve the quality of life. Due to the complexity of ED pathophysiology, combination therapy may be more effective, such as oral medications and physical therapy. Moreover, exosomes have some applicative advantages. First, exosomes can be stored for a long time at low temperature, which is convenient for storage and transportation (84). Secondly, the protein, nucleic acid and other contents in exosomes are encapsulated by lipids, which is the structural basis for good stability (85). It is important to note that exosomes may circumvent many issues related to ethics. The exosome may be the future of ED treatment, nevertheless research is still at the preliminary stage. Basic researches and clinical trials of exosomes in the treatment of ED lay a solid foundation for the clinical translation of exosome therapy.

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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