Hypothesis / aims of study
Stress urinary incontinence (SUI) is a common and annoying medical condition affecting millions of women around the world. Despite the common use of mid-urethral sling surgery, this procedure is associated with a high level of complications, and therefore, there is a need for new medical treatment options. Stem cell-based therapy has gained attention as a promising treatment for SUI. Meanwhile, an increasing number of studies demonstrate that the paracrine effect rather than the stem cell differentiation is the main factor resulting the therapeutic effect. Exosomes are 40- to 150-nm-sized nanoparticles originating from multivesicular bodies (MVBs) and important paracrine effectors in intercellular communication. They function by transferring proteins and genetic materials to target cells. Recent studies have reported that exosomes secreted by adipose tissue-derived stem cells (ADSCs) show potential in tissue regeneration and protection, including bone, muscle, skin, and brain tissue, which support that theory. We hypothesized that ADSCs-derived exosomes could be effective in treating SUI by promoting tissue regeneration after injury. Here, we utilized exosomes derived from human ADSCs and investigate whether it can facilitate recovery after SUI by in vitro and in vivo studies.
Study design, materials and methods
For the in vitro study, a Cell Counting Kit-8 (CCK-8) array and proteomic analysis were performed. CCK-8 array was intervened with control culture medium and culture medium containing different concentrations of exosomes (0.05, 0.5, and 5 µg/ml) for 72 hours. The exosome proteins were determined by a high-performance liquid chromatography (HPLC) system and then the data were conducted by GO analysis and KEGG pathway analysis. For the in vivo study, female rats were divided into four groups: sham, SUI, ADSC and exosomes (n = 12 each). The SUI model was generated by pudendal nerve transection (PNT) and vagina dilation (VD). One hour after the establishment of the animal model, vehicle, hADSCs, and exosomes were respectively injected into the peripheral urethra. After 2, 4, and 8 weeks, the rats underwent cystometry (CMG) and leak point pressure (LPP) testing, and tissues were harvested for histology and immunofluorescence analyses.
The CCK-8 experiment demonstrated that ADSC-derived exosomes could significantly enhance the growth of skeletal muscle and Schwann cell lines in a dose-dependent manner compare to control medium (P<0.05). The proteomics analysis of the exosome identified 1466 proteins that are implicated in various cell functions and pathways. KEGG pathway analysis found two hundred twenty-two different pathways were linked to the exosomes. Some of these proteins are associated with the PI3K-Akt, Jak-STAT, and Wnt pathways that are related to skeletal muscle and nerve regeneration and proliferation. For CMG and LPP testing, bladder capacity (BC) and LPP remained stable at 2, 4, and 8 weeks post-injection in the sham and SUI groups. In the hADSC and exosome groups, the BC and LPP values increased gradually at 2, 4, and 8 weeks post-injection. At each time point, BCs and LPPs of both groups were statistically significant compared with the SUI group (P < 0.05). Histological study showed that the proportion of striated muscle in the urethra of SUI group was significantly decreased compared to that in the sham group. In the hADSC and exosome groups, the proportion of striated muscle increased continuously at 2, 4, and 8 weeks post-injection. At each time point, the proportion of striated muscle of both groups were statistically significant compared with the SUI group (P < 0.05). IF study showed that striated muscle and peripheral nerves of SUI rats were significantly fewer than those of the sham group after eight weeks of injection,. On the contrary, the ADSC and Exosome groups showed similar amount of striated muscle and peripheral nerves compared to the sham group.
Interpretation of results
In vitro studies showed that hADSCs-derived exosomes could significantly enhance the growth of skeletal muscle and Schwann cell lines, and is likely to affect through pathways such as PI3K-Akt, Jak-STAT, and Wnt pathways. In vivo experiments illustrated that exosome derived from hADSCs could improve the urethral function and promote regeneration of striated muscle fiber and peripheral nerve fiber in the urethra of SUI rats. Thus, it is assumed that hADSCs-derived exosomes could improve both functional and histological recovery after SUI.