A total of 11 published preclinical studies were included in our analysis. Overall, our analysis suggests that stem cell derived exosomes could ameliorate erectile dysfunction and structural changes in various types of ED models.
Penile erection is a series of vascular event closely related to the endothelium and smooth muscle cells of corpus cavernosum, which histologically form the basic structure of sinusoids. When the smooth muscle is contracted, the blood inflow through the cavernous artery limitedly, but blood outflows through the subtunical venular plexus freely, thus resulting the flaccid state of penis.31 Upon sexual stimulation, non-adrenergic non-cholinergic (NANC) nerve fibers release nitric oxide (NO), which activates guanylyl cyclase to increase the concentration of cyclic guanosine monophosphate (cGMP). Besides, acetylcholine released from parasympathetic cholinergic nerve fibers causes activation of adenylyl cyclase, increasing the concentration of cyclic adenosine monophosphate (cAMP). The high level of cGMP and cAMP decrease intracellular Ca2 + levels and lead to smooth muscle cell relaxation, followed by normal erection. If any of these processes is interrupted, erectile dysfunction may happen. For example, cavernous nerves injury causes downregulation in nerve signaling of the corpora cavernosa, which reduces NO level in smooth muscle, increases apoptosis in both smooth muscle and endothelium of blood vessels, and upregulates fibro-genetic cytokines to form collagenization of the smooth muscle. These functional and structural changes lead to Veno-occlusive dysfunction32–34. Hypoxia can cause a decrease in prostaglandin E1 levels of corpora cavernosa, which commonly inhibit pro-fibrotic cytokines, such as TGFβ1.35,36 These pro-fibrotic cytokines enhance collagen deposition, decrease the smooth muscle content, reduce elasticity of the penis, and impair the ability of the cavernosa to compress the subtunical veins, causing veno-occlusive dysfunction32. As reported, the mechanisms of diabetic ED observed in rat models may include elevated glycation end-products and oxygen free radical levels, impaired synthesis of nNOS, and decreased cGMP- dependent kinase-137,38. In a word, ED is a multifactorial condition with a complex neurovascular process, which has been shown to be strongly associated with the loss and dysfunction of the corporal endothelium and smooth muscle.
Clinically, refractory male erectile dysfunction shows resistance to drug therapy, which has little effect on them. Facing this obstacle, stem cell therapy was recognized as a promising novel method in ED treatment and considerable studies have proved its feasibility in both animal models and clinical trials, mostly having significant effects39,40.
Stem cells derived from embryonic or adult tissues, have the capability of self-renewal, proliferation and multipotential differentiation. The regenerative properties have been established in tissue engineering and regenerative medicine researches.41,42
Recently, some studies considered that the beneficial effects of transplanted stem cells could not be merely explained by engraftment or differentiation into specific cells.43 Scientists have paid more attention to the paracrine secretion of stem cells, including chemoattractant molecules, bioactive factors and EVs,44,45 which play a key role in the paracrine secretion. Exosomes are 50–100 nm membrane-bound EVs, in which content varies dependent on the original cells and the activation status, including non-coding small RNAs, mRNAs, proteins, and lipids46. Exosomes have been proved to serve multiple physiologic and pathologic functions via regulating intercellular communication. The therapeutic potential of exosomes in different diseases is a hot topic to date. Lai et al47. identified exosomes derived from mesenchymal stem cells (MSCs) exerted protective effect on cardiac tissue following myocardial infarction (MI). Zhang et al48. demonstrate that MSCs derived exosomes effectively promoted functional recovery in rats after traumatic brain injury by facilitating endogenous angiogenesis and neurogenesis.
Corresponding to previous stem cell therapy studies, our study revealed that stem cell derived exosomes increased the smooth muscle to collagen ratio, and the expression of α-SMA, CD31, nNOS and eNOS damaged by ED. CD31 can be considered biomarker of endothelium contents49, while a-SMA and SM/Collagen indicated the smooth muscle contents in the corpus cavernosum of rats. This meant exosomes can improve corpus cavernosum tissue structure to ameliorate erectile function and then the therapeutic efficacy can last for a longer time. Besides, our study proved the downregulation expression level of TGF-β1 and caspase3. As a kind of profibrotic cytokine, TGFβ1was recognized as a key factor related to the formation and development of corporal fibrosis such as in PD.50 Kim et al reported that the activation of TGF-β1 signaling initiated collagen accumulation and deposition.51 Activation of caspases was recognized as the biochemical marker for apoptosis and detection of caspase 3 was widely used in apoptotic signals examination research52. Vasculogenic ED induced by artery injury was characterized as the ischemic and hypoxic state of corpus cavernosum, which may increase the release of reactive oxygen species (ROS), leading to cell apoptosis53,54. It is reported that oxidative stress is an important factor of ED progress for penile ischemia. Liu et al55 found that cell apoptosis can be induced by ROS, which also increased cytoprotective autophagy, slightly reducing apoptosis. In our study, the administration of stem cell derived exosomes decreased the expression level of caspase 3 and TGF-β1, which indicated that exosomes possess the ability to inhibit fibrosis and apoptosis, ensuring the functional endothelium and smooth muscle contents in corpus cavernosum. Our study also demonstrated that stem cells derived exosomes can make functional changes of corpus cavernosum via the NO/cGMP signaling pathway, 31which was the most important mechanism known to regulate erection. The eNOS synthesized by endothelial cells and nNOS56 in cavernous nerves both significantly increased after exosomes therapy in our study. The outcome was consistent with ICP/MAP analysis.
In the included studies, only two studies used exosomes generated from human urine-derived stem cells, 28,29the others used exosomes derived from ADSCs or BMSCs. They showed no difference to the therapeutic efficacy in our research. Although exosomes can be generated by most cells, the exosomes derived from MSCs were used in most researches to treat ED. MSCs were more widely used than other stem cells in researches for the superiority of abundant tissue source and easy isolation. MSCs can be isolated from several tissues, including bone marrow, adipose tissue, Wharton’s Jelly (WJ) tissue, umbilical cord blood, and neonatal teeth.57 Furthermore, ADSC and BMSC were exploited mostly. Non-coding RNAs, such as miRNA, snoRNA, tRNA, enriched in exosomes, may exert important biological functions, conveying properties of parental cells. For example, tRNAs accounted over 50% proportion of total small RNAs in exosomes derived from adipose-derived stem cells, while represented 23–25% in BMSC derived exosomes. Besides, specific tRNAs were abundant in exosomes compared to the cell. Interestingly, miRNAs were the major content of the cellular small RNA in MSCs, and the discrepancy may suggest preferential sorting and release.46,58 Exosomes may exhibit heterogeneity of content even originated from the same parental cells.59–61 Both the subcellular origin and cell activation status were responsible for molecular heterogeneity of exosomes.62,63 Because of the limitation of exosome isolation methods, bulk isolates rather than pure exosome population isolates were used in majority of studies when evaluating their therapeutic efficacy.46
Exosomes isolated from urine also contained substantial non- coding small RNAs, such as tRNA and rRNA, while the exact functions need further study. In fact, the researches on exosome-mediated communication mostly focused on well-known RNA species such as miRNAs and mRNAs, for the challenges on detection sensitivity and specificity of exosomes contents.46 Zhu et al25 found that ADSC derived exosomes contained some microRNAs with proangiogenic (miR-126, miR-130a and miR-132) and antifibrotic (miR-let7b and miR-let7c) functions. Besides the membrane proteins of exosomes influencing the interaction with recipient cells, there were functional proteins intra exosomes involving intracellular signaling mediation. Wang et al23 used the transmembrane serine protease Corin in ADSC derived exosomes to improve erectile dysfunction in diabetic rats and suggested that it may play a role through the ANP/NO/cGMP signaling pathway.
Compared with stem cell therapy, exosomes have many advantages, including (i) greater stability and ease of storage and management, (ii) preclusion the risk of tumor formation, and (iii) a lower likelihood of an immune rejection.14,21
IC injection was the main method to administrate cells or exosomes into the cavernous body in most researches. However, in terms of histologically high vascularization, effective retention of exosome or stem cells in local tissue is a key challenge. Exosomes derived from mature body cells may have the superiority of better histocompatibility, compared with that derived from stem cells. Song et al27 thought that exosomes generated from corpus cavernosum smooth muscle cells (CCSMCs) were more easily taken up and retained in the corpus cavernosum of diabetic ED rats than those from BMSC or ADSC.
In this study, we analyzed the efficiency of exosome therapy for ED using a meta-analysis to obtain a powerful conclusion. To the best of our knowledge, this is the first meta-analysis providing comprehensive insights into the effects of stem cell derived exosomes on ED in rats. The value of systematic review of experimental animal studies has been steadily understood.64,65 The consistent results of exosome therapy efficacy across various ED models in our study would provide reassurance that human beings might respond in the same way. While, here are still several limitations in this study. High degree of heterogeneity remains in ICP/MAP outcome after subgroup analysis. This may be due to the methodological heterogeneity of the included studies, as exosome types, extraction methods and animal models used in each study were quite different. In view of the limited number of included studies, more accurate subgroup analysis cannot be performed at present. Besides, different software (e. g. SPSS, GraphPad Prism and Stata) are applied in included studies, which may cause high statistical heterogeneity.
The egger test shows publication bias in our study, which may affect the credibility of the conclusions. However, we have tried our best to retrieve animal intervention studies on exosome treatment for ED, including preprint database like bioRxiv and medRxiv, but failing to find more relevant researches. This indicates that exosome therapy for ED is a relatively new topic, and more experimental data are needed to support its effectiveness.
However, in the meta-analysis of structural changes, low heterogeneity of SM/ Collagen, CD31, and eNOS outcomes guaranteed the credibility of the conclusion that exosome treatment ameliorated the cavernosum structure, and verified the ICP/MAP functional recovery from the side.