Table 1.
QPCR oligonucleotides.
Fig 1.
Wound healing of human bladder smooth muscle cell (BSMC) monolayers increases the regenerative pathway, YAP/WWTR1, and transcription of downstream targets.
As a cognate for wound healing during initial stages of spontaneous regeneration, scratch wounds were created in confluent monolayers of quiescent BSMC. A, B: In vitro scratch wound induces YAP/WWTR1 signaling and downstream target genes. Scratch wound assay of cultured bladder SMC caused increased intensity of nuclear YAP in migrating cells on the wound edge at 6 hours. n = 4. C: Representative images of migrating bladder SMC after scratch wound time-lapse microscopy with and without verteporfin (VP), an inhibitor of YAP/WWTR1 activity (Supporting information S1 Video shows time lapse micrography of the SMC migration). D: YAP and WWTR1 mRNA expression levels were increased by scratch wound healing. VP lowered the WWTR1 expression induced by wound healing. MST1 expression decreased after scratch wounding. E: Scratch wound of BSMC increases expression of downstream target genes of YAP & WWTR1 signaling, BDNF, CYR61 and CTGF. The inhibitor of Verteporfin (VP, 0.1 μM) significantly reduced scratch wound-induced expression of CYR61 and BDNF. *, p <0.05; **, p<0.01; ***, p<0.005.
Fig 2.
Spontaneous regeneration occurs rapidly after subtotal cystectomy (STC).
One week post-STC, bladder mass (A) is lower than sham or cystotomy bladders, but STC bladders are higher than t = 0 partial cystectomy bladders (dotted line). Dotted line indicates mean size of STC bladders cut at the same levels as other STC bladders that were removed and weighed on day 0. A: After 1+7 weeks, the STC bladder mass is lower than sham or cystotomy bladders, but is still higher than the time = 0 STC control (dashed line). B: Bladder to body weight ratios decreased in STC, while incision of the bladder, as a control for wound healing without cystectomy, lead to increased bladder/body weight ratio at 1 week, but not 7 weeks after incision. C: Residual urine volumes are significantly less in week1 STC, but regain lost residual volume by week7 STC. D: Immunofluorescent staining for WWTR1 and Collagen I in sham, STC, cystotomy and PBO treatments, at 1 and 7 weeks. PBO was performed by partial closure of the peri-urethra in Sprague-Dawley female rats. Collagen deposition is dysregulated in both STC and cystotomy treatments, with a significant increase in WWTR1 expression in cystotomy treatments at 1 week. WWTR1 expression at 7 weeks is slightly increased in incision treatments, with an even slighter increase in expression + collagen dysregulation upon PBO which represents SMC hypertrophy. E: Immunofluorescent staining for YAP and Calponin1 (CNN1) in sham, STC, cystotomy, and PBO treatment, at 1 and 7 weeks. YAP expression is similar at 1 and 7 weeks across all treatments. However, STC treatment at 1 week shows SMC hypertrophy, and even increased CNN1 dysregulation in cystotomy treatments. PBO treatment demonstrates slightly decreased expression of CNN1, with maintained SMC hypertrophy at 7 weeks. *, p <0.05; **, p<0.01; ***, p<0.005.
Fig 3.
STC in the bladder leads to increased expression of YAP/WWTR1 target genes.
A: Pan-BDNF at 1 and 7 weeks augmented significantly with STC, but not incision at 1week. B, C: Isoforms of BDNF shows differential regulation in the different injury models. While Variant 1 (exon VI) was not significantly increased during STC (B), significant elevation of the Variant 5 (exon IV) isoform was seen during STC (C). D: CTGF mRNA expression was not increased significantly by STC, though incision was significantly lower than STC. E: CYR61 mRNA expression increased during STC at 1 week, with a trend at 7 weeks. Incision alone induced minimal change. F: NTRK2 mRNA increased initially at 1 week STC, but decreased by 7 weeks STC. The incision control showed the opposite trend. *, p <0.05; **, p<0.01; ***, p<0.005.
Fig 4.
Nerve fibre density in regenerating bladders was increased in regions surrounding smooth muscle bundles (A) and near the basement membrane and mucosa (B). Staining with β3-tubulin antibody (deep blue) demonstrated significantly altered presence of peripheral nerve fibres in the bladders. Smooth muscle was immunostained with calponin (red). (C) Quantification of nerve densities in A and B. *, p<0.05.
Fig 5.
Migration during scratch wound healing depends on YAP/WWTR1 signaling and BDNF.
A: Using live imaging of the scratch wound edges, migration of bladder smooth muscle cells was assessed over 12 hours, with and without inhibitors (as in Fig 1C, S1 Video). % Closure was calculated by comparing the change in wound area. Wound closure occurred rapidly after initiation, with 68% closure at 7 hours and 85% closure at 12 hours. Soluble BDNF receptor (NTRK2) was able to reduce wound closure at both 7 and 12 hours. Verteporfin (0.1uM) reduced wound closure at 7 hours. B: GNF 5837, an inhibitor of the NTRK2 kinase, caused a significant decrease in bladder SMC migration after scratch wounding, reducing closure by >3-fold. C: BDNF protein in scratched BSMC lysates was upregulated during scratch wounds, by ELISA. VP inhibited production of BDNF protein. D: Scratch wound was performed with and without VP plus/minus exogenous CTGF, CYR61 and BDNF proteins. BDNF increased migration above vehicle levels, but demonstrated a greater increase relative to the VP-treated scratch wounds which had lower migration levels. n = 4 wells were performed for each scratch wound test, with 4–6 fields of view replicates for each n, with student’s t-tests p<0.05 considered significant. * p<0.05, **, p<0.01.
Fig 6.
Schematic of the YAP/WWTR1 signaling pathway responsible for changes after subtotal cystectomy.
Subtotal cystectomy and wound healing induces YAP-dependent BDNF and CYR61 expression. Blocking soluble BDNF decreased migration during scratch wound healing. Pathways to regeneration may also include growth and differentiation pathways that are modulated by BDNF, and CYR61 in smooth muscle cells [33].