Hypothesis / aims of study
Stress urinary incontinence (SUI) and pelvic organ prolapse (POP) are common worldwide problems that affect the quality of life of millions of women. There are many factors contributing to the development of these disorders such as genetic and ethnicity, age, parity, and other health issues. Situations such as pregnancy, vaginal delivery, overweight and age are the risk factors associated to develop stress urinary incontinence (SUI). Pelvic organ prolapse (POP) was detected in 50% of women with urinary incontinence. There is an obvious need to determine whether there are differences in the etiologies of these two pathologies that are the most common pelvic floor disorders, in order to discover novel therapies, as well as to focus on the preventing factors. Pelvic floor tissues of women with SUI are damaged, at an anatomical, cellular and molecular level. Tissue repair normally occurs very quickly after a trauma, which causes tissue injury. Activated fibroblasts, termed myofibroblasts, respond to damage and play a crucial role in tissue repair. A myofibroblast is a cell with a phenotype between a fibroblast and a smooth muscle cell. Presently, it is accepted that myofibroblast are fibroblast-like cells that express α-smooth muscle actin (α-SMA), the actin isoform present in typical contractile smooth muscle cells. These myofibroblasts have contractile activity directly related to the presence of α-SMA. These cells are then capable of initiating wound repair by contracting the edges of the wound. Otherwise, desmin is a muscle-specific protein and a key subunit of the intermediate filament in cardiac, skeletal and smooth muscles. Desmin play a critical role in the maintenance of structural and mechanical integrity of the contractile apparatus in muscle tissues. Our aim was to isolate and characterize fibroblast cells from the suburethral mucosa of patients with stress urinary incontinence (SUI) and pelvic organ prolapse (POP) without incontinence and analyze the existence of differences between them.
Study design, materials and methods
Suburethral tissues were obtained from patients with SUI or POP without incontinence under informed consent and cell cultures were established by enzymatic digestion. Isolated cells were cultured in DMEM supplemented with 10% fetal bovine serum. The expression of α-SMA and desmin genes was analyzed by quantitative real-time PCR during the expansion culture. The expression of both proteins was analyzed by immunofluorescence or alphaLISA techniques. Desmin protein was also analyzed in paraffin embedded suburethral tissues by immunocytochemistry. Cell contractibility was measured by CytoSelect™ 24-well Cell Contraction Assay Kit according to the manufacturer instructions.
The cells obtained from both patients have a fibroblast-like morphology. The results showed that the expression of α-SMA gene is greater in SUI cell cultures compared to POP cultures and maintained during the expansion culture. In addition, a higher protein expression of α-SMA was observed by immunofluorescence in isolated cells from SUI patients. Basal levels of desmin gene was lower in SUI cells respect to POP cells. However, during the expansion culture the desmin gene is increased in SUI cells until the end of expansion culture while is decreased in POP cells. Furthermore, desmin protein has the same pattern when analyzed by alphaLISA. What is more, histological analysis of suburethral tissues by immunocytochemistry for desmin confirmed the lower levels of this protein in tissues of patients with SUI.
Wound healing is comprised of three processes: epithelialization, connective tissue deposition, and contraction. The contraction process is believed to be mediated by specialized fibroblasts called myofibroblasts. To assess cell contractibility in vitro and screen cell contraction differences between SUI and POP cells without incontinence we used the collagen gel contraction assay. The results showed that SUI cells have higher contraction capability respect to POP cells.
Interpretation of results
Cells isolated from suburethral mucosa of women with stress urinary incontinence are myofibroblasts with contractile capacity and different to the cells isolated from suburethral mucosa of women pelvic organ prolapse that seem to be a less activated cell type, such as the fibroblast.