Bone marrow stem cells secretome accelerates simulated birth trauma-induced stress urinary incontinence recovery in rats

Abstract

Stress urinary incontinence (SUI) is defined as involuntary urine leakage during physical activities that increase the intra-abdominal pressure on the bladder. We studied bone marrow stem cell (BMSC) secretome-induced activation of anterior vaginal wall (AVW) fibroblasts and its ability to accelerate SUI recovery following vaginal distention (VD) in a rat model of birth trauma using BMSC-conditioned medium (BMSC-CM) and concentrated conditioned medium (CCM). BMSC-CM enhanced the proliferation, migration, and collagen synthesizing abilities of fibroblasts. Differentially expressed genes in BMSC-CM-induced fibroblasts were mainly enriched for cell adhesion, extracellular fibril organization and angiogenesis. Treatment with the JAK2 inhibitor AG490 reversed BMSC-CM-induced activation of the JAK2/STAT4 pathway. Periurethral injection with BMSC-CCM markedly enhanced the abdominal leak point pressure (LPP) in rats after VD. Histological analysis revealed increased numbers of fibroblasts, improved collagen fibers arrangement and elevated collagens content in the AVW of rats receiving BMSC-CCM. These findings suggest the BMSC secretome activates AVW fibroblasts and contributes to the functional and anatomic recovery of simulated birth trauma-induced SUI in rats.

Keywords: JAK2/STAT4; bone marrow stem cells; fibroblasts; secretome; stress urinary incontinence.

Figure 1

BMSC-CM enhanced the proliferation and migration ability of fibroblasts in vitro. (A) Positive staining of vimentin and negative staining of α-SMA in cultured fibroblasts by immunofluorescence (magnification, ×200). Scale bar = 200 μm. (B) Wound–scratch assay in fibroblasts treated with different media at 0 h, 24 h, 48 h (magnification, ×40). Scale bar = 400μm. (C) The percentage of migration area in different groups. (D) Cell number displayed as OD value of fibroblasts treated with different media in the CCK-8 assay. Data are shown as means ± standard deviation (SD). ^*P < 0.05; ^**P < 0.01; ^***P < 0.001. BMSC-CM, bone marrow stem cell-conditioned medium; con, control medium treatment group; CM, BMSC-CM treatment group.

Figure 2

BMSC-CM promoted collagen production in fibroblasts. (A) RT-PCR showing relative mRNA expression of collagen type I alpha 1 chain (COL1A1), collagen type I alpha 2 chain (COL1A2), collagen type III alpha 1 chain (COL3A1), matrix metalloproteinases MMP1, MMP2, and MMP3 in fibroblasts treated with different media (versus GAPDH mRNA). (B) Western blotting showing protein levels of collagen I, collagen III, MMP1, and MMP2 in the two groups of fibroblasts. (C) Relative collagen I, collagen III, MMP1 and MMP2 protein levels (versus GAPDH). Data are shown as means ± standard deviation (SD). ^*P < 0.05; ^**P < 0.01; ^***P < 0.001. BMSC-CM, bone marrow stem cell-conditioned medium; con, control medium treatment group; CM, BMSC-CM treatment group.

Figure 3

RNA-seq revealed the whole genome expression changes in fibroblasts after treatment with BMSC-CM. (A) Volcano plot showing DEGs of BMSC-CM group relative to the control group. The red plot indicates high expression, and the green plot indicates low expression. (B) The upper and lower panels showing heat maps of DEGs in fibroblasts. (C) The GO function analysis indicated DEGs to be enriched in biological processes, cellular components and molecular functions. The node size represented the number of genes enriched in the category. (D) Schematic diagram of possible signal transduction pathways according to RNA-seq results. DEGs, differentially expressed genes; BMSC-CM, bone marrow stem cell-conditioned medium; con, control medium treatment group; CM, BMSC-CM treatment group.

Figure 4

BMSC-CM activated fibroblasts by activating the JAK2/STAT4 pathway. (A) The expression of JAK2, p-JAK2, STAT4, and p-STAT4 in fibroblasts treated with different media was determined by western blotting. (B) The expression of three most significant DEGs (POSTN, COMP, and TGFBI) in fibroblasts treated with JAK2 inhibitor AG490 (10 μM). (C) Wound–scratch assay in fibroblasts treated with or without JAK2 inhibitor AG490 (10 μM) at 0 h, 24 h, and 48 h (magnification, ×40). Scale bar = 400 μm. (D) The percentage of migration area in different groups. (E) The CCK-8 assay showing reduced proliferation of fibroblasts treated with BMSC-CM after treatment with AG490. (F) Collagen synthesis in fibroblasts after AG490 treatment was determined by RT-PCR. Data are shown as means ± standard deviation (SD). ^*P < 0.05; ^**P < 0.01; ^***P < 0.001. BMSC-CM, bone marrow stem cell-conditioned medium; con, control medium treatment group; CM, BMSC-CM treatment group.

Figure 5

BMSC-CCM treatment enhanced LPP of rats under VD. Measurement and quantification of LPP in three rat groups. Data are shown as means ± standard deviation (SD). ^*P < 0.05; ^**P < 0.01; ^***P < 0.001. BMSC-CCM, bone marrow stem cell-concentrated conditioned medium; LPP, leak point pressure; VD, vaginal distention; red arrow: urinary leakage; VD + C, VD + control medium group; VD + CCM, VD + BMSC-CCM group.

Figure 6

BMSC-CCM treatment accelerated the survival of fibroblasts in AVW. The number of vaginal wall fibroblasts in rats was assessed by observing vimentin-positive cells (magnification, ×400). Scale bar = 40 μm. BMSC-CCM, bone marrow stem cell-concentrated conditioned medium; VD, vaginal distention; VD + C, VD + control medium group; VD + CCM, VD + BMSC-CCM group.

Figure 7

BMSC-CCM treatment promoted collagen content in the middle urethra and adjacent AVW tissues of VD rats. (A, D) Masson’s tricolor staining of the middle urethra and adjacent AVW tissues collected from rats. Collagen fibers were stained blue. The images were magnified 40× (scale bar = 200 μm) and 200× (scale bar = 40 μm). (B, E) IHC showing collagen III (stained brownish yellow) expression in tissues (scale bar = 200 μm). (C, F) The expression of desmin and collagen I was determined by western blotting, and the relative collagen I expression (vs. GAPDH) was analyzed. Data are shown as the means ± standard deviation (SD). ^*P < 0.05; ^**P < 0.01; ^***P < 0.001. BMSC-CCM, bone marrow stem cell-concentrated conditioned medium; VD, vaginal distention; EUS, external urethral sphincter; AVW, anterior vaginal wall; USM, urethral smooth muscle; VD + C, VD + control medium group; VD + CCM, VD + BMSC-CCM group.