Small extracellular vesicles secreted by vaginal fibroblasts exert inhibitory effect in female stress urinary incontinence through regulating the function of fibroblasts

Abstract

Stress urinary incontinence (SUI) is a common condition in women and associated with extra-cellular matrix (ECM) reconstruction, which is mainly regulated by fibroblasts. However, the underlying mechanism remains obscure. Small extracellular vesicles (sEVs) play fundamental biological roles in various cellular functions. Some studies suggested that the sEVs were involved in the metabolism of ECM and the function of fibroblasts. The purpose of our study was to investigate the effect of sEVs secreted by vaginal fibroblasts on the pathogenesis of SUI. We showed that the fibroblasts of female anterior vaginal wall secreted sEVs. Moreover, fibroblasts of females with SUI had significantly elevated secretion of sEVs. The collagen contents, proliferation and migration capacity of fibroblasts were decreased when fibroblasts were co-cultured with fibroblasts-derived sEVs (fibroblast-sEVs) from SUI patients. Proteomic analysis revealed that fibroblast-sEVs contained various differentially expressed proteins including TIMP2, TGF-β and ABCC4, which were involved in signaling pathways of fibroblasts regulation. Therefore, we suggested that fibroblast-sEVs contributed to the pathogenesis of SUI through various proteins including TIMP2, TGF-β and ABCC4.

Fig 1. The fibroblast-sEVs from patients with SUI was elevated.

(A and B) Scatter plot distribution of SUI-sEVs and non SUI-sEVs with Nanoparticle tracking analysis measurements. (C) Quantitative comparison results of the number of sEVs in the SUI and non-SUI groups. (D) Semi-quantitative comparison results of total protein content of sEVs in the SUI and non-SUI groups. N: non-SUI group, SUI: SUI group, N = 3 in each group. Data represented means±SD of 3 independent experiments. * t-test P value <0.05.

Fig 2. SUI-sEVs negatively regulated type I and III collagen expression in fibroblasts.

Fibroblasts were incubated with control medium or medium containing SUI-sEVs (S-sEVs) or medium containing non SUI-sEVs (N-sEVs) for 48 h. (A) The expression of type I and III collagen were determined by the Western blot assay. (B) Quantification of type I and III collagen expression. N = 3 in each group. Data represented means±SD of 3 independent experiments. * t-test P value <0.05.

Fig 3. SUI-sEVs decreased the migration ability of primary cultured fibroblasts.

(A) Fibroblasts were undergone scratch test, the wound healing changes were recorded between three groups at 0, 24 and 48h. (B) The variation of scratching space between groups is shown in the line chart. SUI-sEVs significant decreased the migration ability of primary cultured fibroblasts at 48h when compared to the cells in control group and treated with the non SUI-sEVs. N = 3 in each group. Data represented means±SD of 3 independent experiments. * t-test P value <0.05.

Fig 4. SUI-sEVs decreased the proliferation of fibroblasts.

Fibroblasts were cultured with control medium or medium containing SUI-sEVs or medium containing non SUI-sEVs for 24, 48 and 72h, respectively. (A) Cell proliferation was determined with a CCK-8 assay. (B) The percentage of proliferative fibroblasts in three groups. SUI-sEVs significantly decreased the proliferation of fibroblasts after 72h when compared to the cells in control group and treated with the non SUI-sEVs. N = 3 in each group. Data represented means±SD of 3 independent experiments. * t-test P value <0.05.

Fig 5. Summary of significant expressed proteins in fibroblast-sEVs of SUI patients or controls.

(A) The number of identified proteins in each group was presented by Venn diagram. (B) Distribution of differentially regulated proteins. (C) Some significantly upregulated proteins in fibroblast-sEVs of SUI patients. (D) Some significantly downregulated proteins in fibroblast-sEVs of SUI patients. N = 3 in control group and N = 5 in SUI group.

Fig 6. The Bioinformatics analysis of the proteins carried by the sEVs.

(A-C) GO functional classification of fibroblast-sEVs proteomes. (A) Biological process, (B) cellular component, and (C) molecular function. The numbers after comma indicate the count of the proteins. (D) The KEGG analysis of the sEVs.