Spiritosanto, Elisabeth (2020) Understanding bladder smooth muscle repair capacity in healthy and overactive bladders. (PhD thesis), Kingston University, .
Abstract
Overactive bladder (OAB) is a urological condition characterised by frequency, urgency, nocturia and sometimes incontinence. The prevalence of OAB in the USA and Europe is estimated to be 10-17%(Coyne et al., 2008), and it has a majorimpact on the patient’s quality of life, particularly in the elderly. Since the aetiology of the pathology is poorly understood, therapeutic efforts to date are aimed primarily at symptom resolution rather than targeting underlying causes or mechanisms that could improve tissue function. Insulin-like growth factor 1 (IGF-I) stimulates proliferation and differentiation in muscle growth and repair. Alternative splicing generates three distinct variants in human tissues: IGF-1Ea, which is the predominant variant in muscle and liver, IGF-1Eb, and IGF-1Ec. The translated product of IGF-1Ec is also known as “mechano growth factor” (MGF) due to its transcriptional upregulation following injury and mechanical stress. MGF role and mechanism of action have been studied mainly in skeletal muscle, where IGF-1Ec has been shown to be upregulated after muscle damage, correlating with repair and regeneration. On the other hand, very little is known about its function in smooth muscle. The hypothesis that drove this study is that MGF may be produced in normal bladder to promote detrusor cell proliferation and tissue repair. In the OAB, MGF production may be decreased, leading to a reduction in smooth muscle cell proliferation and loss of function. Our aim was to test this hypothesis, investigating MGF function in the bladder. We determined MGF protein expression in bladder tissue by immunohistochemistry, and quantified expression in normal and pathological human bladder biopsies by Western blot and immunohistochemistry techniques. We further assessed the tissue composition of each biopsy sample using RT-qPCR and examined the statistical correlation between clinical parameters, MGF expression and biopsy composition. Finally, we examined the effect of MGF peptide on bladder smooth muscle proliferation in vitro. Our data shows for the first time that MGF is detectable in normal and overactive bladders, and expression is around two-fold higher in pathological (mean relative expression=0.48 ± 0.09, n=28) compared to control samples (mean=0.27 ± 0.07, n=12), though the difference does not reach statistical significance (p=0.11). The biopsy samples showed different composition patterns in terms of presence/absence of the different layers of the bladder. The increase in MGF expression was particularly striking in the subset of the samples expressing markers of smooth muscle tissue (mean=0.49 ± 0.11 in pathological samples and 0.23 ± 0.09 in control samples; p=0.06; n=17 and n=8, respectively). This suggests that smooth-muscle derived MGF increased in patients with OAB, perhapsreflecting activation of tissue regeneration in an attempt to compensate for muscle degeneration associated with the pathology of OAB. While a trend towards a positive correlation between maximum cystometric capacity (MCC) of the bladder and MGF expression was observed, statistical significance was not reached (p=0.15). Finally, the preliminary treatment of primary bladder III cells with different concentrations of MGF peptide showed a significant increase in cellular proliferation following treatment with 100 ng/ml MGF compared to the control after 72 hours, as determined by EdU assay (p=0.008). The results of this study suggest that increased MGF is associated with OAB pathology, and its application may represent a new avenue of research for OAB patients. However, further research is required.
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