DNA Methylation Reduces the Yes-Associated Protein 1/WW Domain Containing Transcription Regulator 1 Pathway and Prevents Pathologic Remodeling during Bladder Obstruction by Limiting Expression of BDNF

Chronic bladder obstruction and bladder smooth muscle cell (SMC) stretch provide fibrotic and mechanical environments that can lead to epigenetic change. Therefore, we examined the role of DNA methylation in bladder pathology and transcriptional control. Sprague-Dawley female rats underwent partial bladder obstruction by ligation of a silk suture around the proximal urethra next to a 0.9-mm steel rod. Sham operation comprised passing the suture around the urethra. After 2 weeks, rats were randomized to normal saline or DNA methyltransferase inhibitor, 5-aza-2-deoxycytidine (DAC) at 1 mg/kg, three times/week intraperitoneally. After 6 weeks, bladders were weighed and divided for histology and RNA analysis by high-throughput real-time quantitative PCR arrays. DAC treatment during obstruction in vivo profoundly augmented brain-derived neurotrophic factor (BDNF) expression compared with the obstruction with vehicle group, which was statistically correlated with pathophysiologic parameters. BDNF, cysteine rich angiogenic inducer 61 (CYR61), and connective tissue growth factor (CTGF) expression clustered tightly together using Pearson's correlation analysis. Their promoters were associated with the TEA domain family member 1 (TEAD1) and Yes-associated protein 1/WW domain containing transcription regulator 1 pathways. Interestingly, DAC treatment increased BDNF expression in bladder SMCs (P < 0.0002). Stretch-induced BDNF was inhibited by the YAP/WWTR1 inhibitor verteporfin. Verteporfin improved the SMC phenotype (proliferative markers and SMC marker expression), in part by reducing BDNF. Expression of BDNF is limited by DNA methylation and associated with pathophysiologic changes during partial bladder outlet obstruction and SMC phenotypic change in vitro. Chronic bladder obstruction and bladder smooth muscle cell (SMC) stretch provide fibrotic and mechanical environments that can lead to epigenetic change. Therefore, we examined the role of DNA methylation in bladder pathology and transcriptional control. Sprague-Dawley female rats underwent partial bladder obstruction by ligation of a silk suture around the proximal urethra next to a 0.9-mm steel rod. Sham operation comprised passing the suture around the urethra. After 2 weeks, rats were randomized to normal saline or DNA methyltransferase inhibitor, 5-aza-2-deoxycytidine (DAC) at 1 mg/kg, three times/week intraperitoneally. After 6 weeks, bladders were weighed and divided for histology and RNA analysis by high-throughput real-time quantitative PCR arrays. DAC treatment during obstruction in vivo profoundly augmented brain-derived neurotrophic factor (BDNF) expression compared with the obstruction with vehicle group, which was statistically correlated with pathophysiologic parameters. BDNF, cysteine rich angiogenic inducer 61 (CYR61), and connective tissue growth factor (CTGF) expression clustered tightly together using Pearson's correlation analysis. Their promoters were associated with the TEA domain family member 1 (TEAD1) and Yes-associated protein 1/WW domain containing transcription regulator 1 pathways. Interestingly, DAC treatment increased BDNF expression in bladder SMCs (P < 0.0002). Stretch-induced BDNF was inhibited by the YAP/WWTR1 inhibitor verteporfin. Verteporfin improved the SMC phenotype (proliferative markers and SMC marker expression), in part by reducing BDNF. Expression of BDNF is limited by DNA methylation and associated with pathophysiologic changes during partial bladder outlet obstruction and SMC phenotypic change in vitro. Partial bladder outlet obstruction (PBO), occurring in diseases such as prostate hyperplasia and urethral valves, is a widespread cause of urinary dysfunction, patient discomfort, and overactive bladder. It was responsible for $65.9 billion in US 2007 health care costs.1Ganz M.L. Smalarz A.M. Krupski T.L. Anger J.T. Hu J.C. Wittrup-Jensen K.U. Pashos C.L. Economic costs of overactive bladder in the United States.Urology. 2010; 75: 526-532.e18Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar Although removing the obstruction partially improves the functional and pathologic sequelae, ongoing symptoms remain in many patients. Epigenetic changes, such as DNA methylation, play an important role in both adaptive and recovery processes, including fibrosis and proliferation.2Tao H. Yang J.-J. Shi K.-H. Deng Z.-Y. Li J. DNA methylation in cardiac fibrosis: new advances and perspectives.Toxicology. 2014; 323: 125-129Crossref PubMed Scopus (31) Google Scholar, 3Jiang J.-X. Aitken K.J. Sotiropoulos C. Sotiropolous C. Kirwan T. Panchal T. Zhang N. Pu S. Wodak S. Tolg C. Bägli D.J. Phenotypic switching induced by damaged matrix is associated with DNA methyltransferase 3A (DNMT3A) activity and nuclear localization in smooth muscle cells (SMC).PLoS One. 2013; 8: e69089Crossref PubMed Scopus (17) Google Scholar Epigenetic mechanisms may be critical components of the response to obstructive stimuli (matrix, hypoxia, inflammatory, and mechanical stimuli). Because DNA methylation may be crucial to the maladaptive or adaptive responses to these stimuli, a hypomethylating agent was used in a rodent model of PBO to evaluate the role of DNA methylation in bladder remodeling and gene expression during obstruction. This invites investigation of potential epigenetic mechanisms in bladder tissue homeostasis and repair. During aging, global loss of DNA methylation occurs across the genome. The aging bladder is commonly clinically associated with dysfunctional changes that include the gradual onset of irritative and obstructive bladder symptoms. However, it is not known if DNA methylation may also contribute to the clinical pathology of the aging bladder. In vitro, DNA methylation changes dynamically in response to obstructive stimuli in bladder smooth muscle cells alongside proliferation.3Jiang J.-X. Aitken K.J. Sotiropoulos C. Sotiropolous C. Kirwan T. Panchal T. Zhang N. Pu S. Wodak S. Tolg C. Bägli D.J. Phenotypic switching induced by damaged matrix is associated with DNA methyltransferase 3A (DNMT3A) activity and nuclear localization in smooth muscle cells (SMC).PLoS One. 2013; 8: e69089Crossref PubMed Scopus (17) Google Scholar In this study, we investigated the effect of DNA methylation during obstruction of nonaging rats to isolate DNA methylation from other age-related pathologies. Functional bladder parameters were assessed using metabolic cages and associated gene expression during obstruction in nonaging rats while inhibiting DNA methylation. By studying candidate gene expression, the most profound and significant changes in gene expression among all groups were identified as brain-derived neurotrophic factor (BDNF) and connective tissue growth factor (CTGF). Using correlation analysis, novel and discrete changes in gene expression in Bdnf and CTGF, relevant to obstructive bladder dysfunction, were identified. It was further determined that BDNF, cysteine rich angiogenic inducer 61 (CYR61), and CTGF share transcriptional regulation through the YAP/WWTR1 pathway. BDNF's massive increase in transcript level and localization around smooth muscle bundles led us to investigate its role more clearly. BDNF can induce highly contrasting effects during bladder obstruction,4Cruz C.D. Neurotrophins in bladder function: what do we know and where do we go from here?.Neurourol Urodyn. 2014; 33: 39-45Crossref PubMed Scopus (52) Google Scholar, 5Song Q.-X. Chermansky C.J. Birder L.A. Li L. Damaser M.S. Brain-derived neurotrophic factor in urinary continence and incontinence.Nat Rev Urol. 2014; 11: 579-588Crossref PubMed Scopus (20) Google Scholar including overactivity, but also restoration of neuronal function. The functional role of BDNF on bladder smooth muscle phenotype has previously not been examined in detail, despite potential implications for regulation of contractile machinery. We examined the role of DNA methylation and YAP/WWTR1 in gene expression and their coordinate effects on bladder smooth muscle cell (BSMC) pathophysiology. Partial bladder outlet obstruction was performed as described,6Schröder A. Kirwan T.P. Jiang J.-X. Aitken K.J. Bägli D.J. Rapamycin attenuates bladder hypertrophy during long-term outlet obstruction in vivo: tissue, matrix and mechanistic insights.J Urol. 2013; 189: 2377-2384Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar under a protocol approved by the Animal Care Committee at The Hospital for Sick Children Research Institute, following policies established in the Canadian Council on Animal Care Guide to the Care and Use of Experimental Animals. In this design, 200 to 250 g Sprague-Dawley female rats (n = 20; Charles River Laboratories, Wilmington, MA) underwent PBO by tying a silk suture around the proximal urethra and a 0.9-mm steel rod; the rod was then removed, leaving the suture behind to form the partial obstruction. Sham operation was performed without tying the suture (n = 12).6Schröder A. Kirwan T.P. Jiang J.-X. Aitken K.J. Bägli D.J. Rapamycin attenuates bladder hypertrophy during long-term outlet obstruction in vivo: tissue, matrix and mechanistic insights.J Urol. 2013; 189: 2377-2384Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar Pain medication was given for the first 24 hours and whenever symptoms of pain were evident. Hypomethylating activity of 5-aza-2'-deoxycytidine (or decitabine; DAC; LC Laboratories, Woburn, MA) treatment was tested by examining Line-1 methylation in the spleens of rats during a separate dose-finding experiment (Supplemental Figure S1). After 2 weeks of obstruction, half of each group was treated with the DNA methyltransferase inhibitor, DAC, at 1 mg/kg, three times/week intraperitoneally for 4 weeks (Supplemental Figure S2). Control animals received normal saline in the same course and route as the treatment.6Schröder A. Kirwan T.P. Jiang J.-X. Aitken K.J. Bägli D.J. Rapamycin attenuates bladder hypertrophy during long-term outlet obstruction in vivo: tissue, matrix and mechanistic insights.J Urol. 2013; 189: 2377-2384Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar Micturition patterns were recorded at 6 weeks 1 to 2 days before sacrifice, using previously described methods.7Sidler M. Aitken K.J. Forward S. Vitkin A. Bägli D.J. Non-invasive voiding assessment in conscious mice.Bladder. 2018; 5: e33Crossref PubMed Google Scholar, 8Sidler M. Aitken K. Jiang J. Bijos D. Belik J. Bägli D.J. Finding NeMO—nerve-sparing mid-urethral obstruction: a pathophysiologically accurate model of rodent partial bladder outlet obstruction.Urology. 2017; 105: 208.e1-208.e9Abstract Full Text Full Text PDF Scopus (6) Google Scholar, 9Sidler M. Aitken K.J. Jiang J.-X. Bägli D.J. Nerve-sparing mid-urethral obstruction (NeMO) in female small rodents.J Vis Exp. 2017; 122: e55288Google Scholar Voiding patterns and volumes (Supplemental Figure S3)8Sidler M. Aitken K. Jiang J. Bijos D. Belik J. Bägli D.J. Finding NeMO—nerve-sparing mid-urethral obstruction: a pathophysiologically accurate model of rodent partial bladder outlet obstruction.Urology. 2017; 105: 208.e1-208.e9Abstract Full Text Full Text PDF Scopus (6) Google Scholar were recorded on a computer using Logger Pro software version 3.9 (Vernier Software & Technology, Beaverton, OR). Residual volumes were measured in anesthetized rats at the time of sacrifice.6Schröder A. Kirwan T.P. Jiang J.-X. Aitken K.J. Bägli D.J. Rapamycin attenuates bladder hypertrophy during long-term outlet obstruction in vivo: tissue, matrix and mechanistic insights.J Urol. 2013; 189: 2377-2384Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar Human BSMCs (Sciencell, Carlsbad, CA) were maintained in BSMC media including growth supplements, fetal bovine serum, and antibiotic/antimycotic (Sciencell). Cells were plated onto collagen I–coated BioFlex plates (Flexcell International, Burlington, NC) at 4 × 104 cells/mL in a 1:1 mix of BSMC medium plus Eagle's minimal essential medium (EMEM) (Wisent, St. Bruno, QC, Canada) plus antibiotic/antimycotic (Wisent). After 24 to 48 hours, cells were serum starved for 1 day in EMEM, then treated in EMEM plus either vehicle (dimethyl sulfoxide), verteporfin (VP; 0.1 μmol/L; MilliporeSigma, Burlington, MA), or decitabine (DAC; 2 μmol/L), 1 hour. Cells were mechanically strained to 5% static elongation on the Flexcell 3000 (Flexcell International) for 6, 8, or 24 hours, using a ramping pattern, as previously described.10Chen K.W. Chen L. Epigenetic regulation of BDNF gene during development and diseases.Int J Mol Sci. 2017; 18 (pii:E571)Google Scholar, 11Aitken K.J. Tolg C. Panchal T. Leslie B. Yu J. Elkelini M. Sabha N. Tse D.J. Lorenzo A.J. Hassouna M. Bägli D.J. Mammalian target of rapamycin (mTOR) induces proliferation and de-differentiation responses to three coordinate pathophysiologic stimuli (mechanical strain, hypoxia, and extracellular matrix remodeling) in rat bladder smooth muscle.Am J Pathol. 2010; 176: 304-319Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar For exogenous BDNF, Trk inhibitor GNF 5837 (both from Alamone Labs, Jerusalem, Israel), and verteporfin treatments, human BSMCs were plated on denatured collagen–coated glass coverslips,12Herz D.B. Aitken K. Bägli D.J. Collagen directly stimulates bladder smooth muscle cell growth in vitro: regulation by extracellular regulated mitogen activated protein kinase.J Urol. 2003; 170: 2072-2076Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar and serum starved overnight before treatments. BDNF was added to serum-starved cells at 0, 2.5, 5, or 10 ng/mL in serum-free EMEM. VP or vehicle (dimethyl sulfoxide at same volume) was added to BSMCs. An initial dosage finding study on human BSMCs showed that VP was not cytotoxic at this level, because it did not decrease cell counts or morphology in unstretched cells (data not shown). Neonatal Sprague-Dawley rat bladder domes were minced and digested using collagenase type IV (Elastin Products Company, Owensville, MO). Digests were washed and cells were preplated to remove fibroblasts. Passaging cells with 0.25 mmol/L trypsin removed contaminating urothelial cells. Cells were plated at passage 3, as late passage primary rat SMC loose differentiation markers. Rat neonatal SMCs were serum starved and stretched in a static ramping pattern for 8 hours, as previously described.10Chen K.W. Chen L. Epigenetic regulation of BDNF gene during development and diseases.Int J Mol Sci. 2017; 18 (pii:E571)Google Scholar Collagen gels were assembled using neutralized type I collagen (2.4 mg/mL; Elastin Products Company, Owensville, MO) and plating into 24-well plates. Serum-starved cells that had been pretreated with vehicle or 10 ng/mL BDNF for 2 days were plated at 5 × 103 cells/mL onto collagen gels in EMEM. After 2 hours to allow for cell attachment, cells were then treated with vehicle, 10 ng/mL BDNF, or BDNF + GNF 5837 (10 μmol/L), then gels were released from the sidewall of the 24-well plate for another 2, 4, and 18 hours. Bladder tissues were harvested, treated in RNAlater at 4°C for 48 hours, dried on sterile gauze, and stored at −80°C until RNA isolation. Tissue samples were crushed under liquid nitrogen, then homogenized at 4°C in Trizol (Thermo Fisher, Waltham, MA) using a refrigerated Bullet Blender (Next Advance, Troy, NY) with steel 0.3 to 1.5 mmol/L pellets for 3 minutes at setting 8. After the steel pellets settled by gravity, the supernatant was processed for RNA isolation.6Schröder A. Kirwan T.P. Jiang J.-X. Aitken K.J. Bägli D.J. Rapamycin attenuates bladder hypertrophy during long-term outlet obstruction in vivo: tissue, matrix and mechanistic insights.J Urol. 2013; 189: 2377-2384Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar Samples from cell culture experiments were isolated by adding Trizol to wells and proceeding as previously described.11Aitken K.J. Tolg C. Panchal T. Leslie B. Yu J. Elkelini M. Sabha N. Tse D.J. Lorenzo A.J. Hassouna M. Bägli D.J. Mammalian target of rapamycin (mTOR) induces proliferation and de-differentiation responses to three coordinate pathophysiologic stimuli (mechanical strain, hypoxia, and extracellular matrix remodeling) in rat bladder smooth muscle.Am J Pathol. 2010; 176: 304-319Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar Each RNA sample from groups PBO, PBO + DAC, and sham (n = 4) was reverse transcribed using Quant-it (Qiagen, Hilden, Germany) and assessed by high-throughput real-time quantitative PCR (HT-qPCR) with a custom array using built-in quality checks. The 85 candidate genes were selected through in silico curation of literature on bladder obstruction and bladder smooth muscle dedifferentiation (Supplemental Table S1). The HTqPCR package on R was used to identify statistical significance of the custom array by limma and t-test.13Dvinge H. Bertone P. HTqPCR: high-throughput analysis and visualization of quantitative real-time PCR data in R.Bioinformatics. 2009; 25: 3325-3326Crossref PubMed Scopus (182) Google Scholar Five reference genes, Rpl13a, B2M, Hprt1, Ldha, and Rplp1, plus β-actin were assessed for optimal normalization on Excel (Microsoft Corp., Redmond, WA) and then the ΔΔCt method. Confirmation of significant HT-qPCR results was performed on a larger set of RNA samples by real-time quantitative PCR of cDNA produced by reverse transcription with Superscript III and Oligo d(t) (Thermo Fisher) and PCR amplification with iQ SYBR Green Supermix (BioRad, Hercules, CA).3Jiang J.-X. Aitken K.J. Sotiropoulos C. Sotiropolous C. Kirwan T. Panchal T. Zhang N. Pu S. Wodak S. Tolg C. Bägli D.J. Phenotypic switching induced by damaged matrix is associated with DNA methyltransferase 3A (DNMT3A) activity and nuclear localization in smooth muscle cells (SMC).PLoS One. 2013; 8: e69089Crossref PubMed Scopus (17) Google Scholar, 14Tolg C. Ahsan A. Dworski S. Kirwan T. Yu J. Aitken K. Bägli D.J. Pathologic bladder microenvironment attenuates smooth muscle differentiation of skin derived precursor cells: implications for tissue regeneration.PLoS One. 2013; 8: e59413Crossref PubMed Scopus (7) Google Scholar Primers are defined in Table 1. Total Ntrk2 expression of neonatal rat BSMC obstructed and sham bladders was assayed using rat Ntrk2 primers from Qiagen. Analysis of variance and post hoc t-tests were performed to identify significant changes in gene expression.Table 1qPCR Oligonucleotide SequencesTargetForwardReverseAmplicon size, bpHuman BDNF promoter gDNA (ChIP primer in gene desert)5′-AGCCCAACAACTTTCCCTTT-3′5′-CGGGGCTGTTAACTCACATT-3′162Human BDNF promoter gDNA (ChIP primer)5′-AGATCACAGAGCCTGCCAGT-3′5′-AATTTGTGTGAGGGGTCTCG-3′177Human BDNF promoter gDNA at EGR1 site (ChIP primer)5′-AGCCTTTCGGGTTCTCATTT-3′5′-AGGTCCAGGGACTCCAAGTT-3′196Human BDNF cDNA5′-TAACGGCGGCAGACAAAAAGA-3′5′-GAAGTATTGCTTCAGTTGGCCT-3′101Human and rat CTGF cDNA5′-AAGACCTGTGGGATGGGC-3′5′-TGGTGCAGCCAGAAAGCTC-3′194Human CYR61 cDNA5′-CGAGGTGGAGTTGACGAGAAA-3′5′-CTTTGAGCACTGGGACCATGA-3′159Human YAP cDNA5′-ACCCTCGTTTTGCCATGAAC-3′5′-TGTGCTGGGATTGATATTCCGTA-3′221Human TAZ/WWTR1 cDNA5′-CCATCACTAATAATAGCTCAGATC-3′5′-GTGATTACAGCCAGGTTAGAAAG-3′338Human β-actin cDNA5′-CGGCTACAGCTTCACCACCA-3′5′-CGGGCAGCTCGTAGCTCTTC-3′140Human B2M cDNA5′-AGGCTATCCAGCGTACTCCA-3′5′-TCATCCAATCCAAATGCGGC-3′348Human GAPDH cDNA5′-GTCAGTGGTGGACCTGACCT-3′5′-TGCTGTAGCCAAATTCGTTG-3′245Human HPRT cDNA5′-GGCGAACCTCTCGGCTTT-3′5′-CATCACTAATCACGACGCCA-3′159Human SDHA cDNA5′-ACTGTTGCAGCACAGCTAGAA-3′5′-TTATGCGATGGATGGACCG-3′283Human UBC cDNA5′-TTAGGACGGGACTTGGGTGA-3′5′-TCACGAAGATCTGCATTGTCAAG-3′240Rat total (pan) Bdnf cDNA5′-TGGGGAGCTGAGCGTGTGTGA-3′5′-TGTGACCGTCCCGCCAGACAT-3′90Rat CYR61 cDNA5′-CTTCCTGTCTTTGGCACGGA-3′5′-AACTCGTGTGGAGATGCCAG-3′134Rat Tead1 cDNA5′-TTTGTGCAGCAGGCCTACCCCATC-3′5′-GGCGAAGCTTGGTTGTGCCAATGGA-3′129Rat Tead4 cDNA5′-ACAGTGACCCCTACCTCGAA-3′5′-GGTCTGCCCAGAACTTCACA-3′135Rat Wwtr1 cDNA5′-GAGGAAGGGCTCGCTTTTGT -3′5′-ATGTTGACCTCGGGACTTTGG-3′88Rat Yap cDNA5′-AATATCAATCCCAGCACAGCA-3′5′-CATTCTGAGTCCCTCCATCC-3′109Rat Ntrk2 cDNA (TrkB T1)5′-GAGCATCTCTCGGTCTATGC-3′5′-CCCATCCAGGGGGATCTTAT-3′146Rat Ntrk2 cDNA (total)QiagenQiagen71Rat β-actin cDNA5′-GGTCGTACCACTGGCATTGTG-3′5′-GCTCGGTCAGGATCTTCATGAG-3′150Rat Gapdh cDNA5′-GATCGTGGAAGGGCTAATGA-3′5′-GAGCTCTGGGATGACTTTGC-3′165Rat Hprt cDNA5′-AGGCCAGACTTTGTTGGATT-3′5′-GCTTTTCCACTTTCGCTGAT-3′118Rat Rpl32 cDNA5′-CATCTGTTTTGCGGCATCA-3′5′-CACCCTGTTGTCGATGCCTC-3′152ChIP, chromatin immunoprecipitation; EGR, early growth response; gDNA, genomic DNA; qPCR, real-time quantitative PCR. Open table in a new tab ChIP, chromatin immunoprecipitation; EGR, early growth response; gDNA, genomic DNA; qPCR, real-time quantitative PCR. To predict transcription factors and pathways of importance for the clustered genes of BDNF, CYR61, and CTGF, the promoters of these genes were analyzed using the web-based programs, TOPGO and OPOSSUM 3.0 (http://opossum.cisreg.ca, last accessed October 31, 2017). Protein extracts from mechanical strain and in vivo obstruction experiments were isolated in radioimmunoprecipitation assay buffer plus protease inhibitor cocktail (Roche, Basel, Switzerland), at 4°C. They were quantitated using the Pierce Bradford Protein quantification kit (Thermo Fisher). Protein samples (10 μg) were electrophoresed on 10% or gradient polyacrylamide gels (BioRad) and transferred to nitrocellulose membranes (Amersham, GE Healthcare, Little Chalfont, UK).11Aitken K.J. Tolg C. Panchal T. Leslie B. Yu J. Elkelini M. Sabha N. Tse D.J. Lorenzo A.J. Hassouna M. Bägli D.J. Mammalian target of rapamycin (mTOR) induces proliferation and de-differentiation responses to three coordinate pathophysiologic stimuli (mechanical strain, hypoxia, and extracellular matrix remodeling) in rat bladder smooth muscle.Am J Pathol. 2010; 176: 304-319Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar Membranes were blocked in skim milk and bovine serum albumin, then probed using antibodies against smooth muscle myosin (rabbit; MilliporeSigma), calponin (mouse; MilliporeSigma), and reference proteins total actin (rabbit; MilliporeSigma) and glyceraldehyde-3-phosphate dehydrogenase (rabbit; Cell Signaling Technology, part of NEB, Ipswich, MA), with secondary anti-rabbit or anti-mouse horseradish peroxidase conjugates (Cell Signaling Technology), as appropriate. Autoradiographs were developed using electrochemiluminescence (Amersham/GE Healthcare). Normalization and quantification were performed on ImageJ version 1.51 (NIH, Bethesda, MD; http://imagej.nih.gov/ij) by analysis of band densities from equivalent band areas on blots for reference proteins compared with target proteins. Bladder tissues were washed in phosphate-buffered saline (PBS) and frozen as for RNA isolation. Protein was isolated and quantitated as for Western blotting in radioimmunoprecipitation assay buffer. Protein lysates (10 μg) and standards were added to the Chemikine BDNF sandwich enzyme-linked immunosorbent assay kit immunoassay plates, following the manufacturer's instructions (R&D Systems, Minneapolis, MN). Cells or 7 μmol/L equatorial bladder cryosections were fixed in paraformaldehyde and washed twice in PBS. Antigen exposure was performed by treatment with 0.2% Triton X-100. Blocking was performed in normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA). Then, staining was performed with the smooth muscle myosin heavy chain rabbit antibody (Medical Group/Alfa Aesar, Tewksbury, MA; or MilliporeSigma), calponin (MilliporeSigma), collagen type I (MilliporeSigma), BDNF (rabbit host; Abclonal, Woburn, MA), YAP, mouse host YAP (Santa Cruz Biotechnology, Dallas, TX), rabbit host YAP/TAZ and phosphorylated YAP (Cell Signaling, a part of NEB), WWTR1 [mouse host (R&D Systems) or rabbit host (Novus Biologicals, Littleton, CO)], and secondary donkey anti–rabbit-Farred Fab fragments or donkey anti-mouse–Cy3 Fab (Jackson ImmunoResearch Laboratories). Nuclei were stained with Hoechst. Slides were mounted with Dako fluorescence mounting medium (Agilent, Santa Clara, CA). Immunofluorescence was captured using an Olympus IX81 microscope (Olympus Corp., Tokyo, Japan) equipped with a Hamamatsu camera (Hamamatsu Photonics K.K, Hamamatsu, Japan) and spectral borealis lasers attached to Volocity software version 6.2 (Perkin Elmer, Waltham, MA). To test for myocyte hypertrophy, transnuclear cross sections of myocytes15Lommatzsch M.1 Braun A. Mannsfeldt A. Botchkarev V.A. Botchkareva N.V. Paus R. Fischer A. Lewin G.R. Renz H. Abundant production of brain-derived neurotrophic factor by adult visceral epithelia. Implications for paracrine and target-derived neurotrophic functions.Am J Pathol. 1999; 155: 1183-1193Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar were measured on Volocity. Intensity of immunofluorescence signals in muscle bundles was evaluated by normalizing the sum of the intensity for each smooth muscle bundle region to the number of cells in each bundle. Immunofluorescence staining for nerves was performed with the β3 tubulin rabbit antibody (MilliporeSigma). Additional processing for both β3 tubulin and YAP antibodies included preclearing treatment with ice-cold methanol for 10 minutes, antigen exposure with 0.3% Triton X-100, blocking in 10% bovine serum albumin (MilliporeSigma) and 10% normal donkey serum (Jackson ImmunoResearch Laboratories), for 30 minutes, and washing steps in 0.1% Triton X-100 in PBS. Equatorial bladder cryosections (7 μm thick) were stained with PicroSirius Red or hematoxylin and eosin. Sections were scanned and imaged using the Aperio system to ascertain the wall thickness at equatorial regions of the bladder, and degree of collagen accumulation by in muscle and nonmuscle tissue compartments.6Schröder A. Kirwan T.P. Jiang J.-X. Aitken K.J. Bägli D.J. Rapamycin attenuates bladder hypertrophy during long-term outlet obstruction in vivo: tissue, matrix and mechanistic insights.J Urol. 2013; 189: 2377-2384Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar Data were included in Pearson's correlations. Chromatin was isolated from human bladder smooth muscle cells, which had been treated with VP or vehicle during stretch or no stretch for 6 hours. Initially, it was found that nuclei could not be efficiently isolated from fixed cells by any kind of homogenization at multiple settings. To maximize isolation of chromatin, it was found that isolation of nuclei was optimized by first washing cells in ice-cold PBS with protease inhibitors, then solubilizing unfixed cell membranes by incubating with 1% Triton X-100 in PBS for exactly 5 minutes on ice. Nuclei were pelleted by centrifugation at 4°C for 5 minutes at 3000 × g. The pelleted nuclei were gently washed at 4°C and fixed in 1% paraformaldehyde in PBS for 10 minutes, after which fixation was immediately blocked with glycine/PBS. Nuclei were washed three times in PBS and placed at −80°C until further processing. Nuclear pellets were sonicated on ice to attain optimal DNA fragmentation (400-bp fragment size). Debris was pelleted at 3000 × g, 3 minutes. Input DNA was isolated from the resulting chromatin in the supernatant by reversing cross-links, phenol extraction, and ethanol precipitation. The chromatin was immunoprecipitated as in the High-Sensitivity ChIP kit (Active Motif, Carlsbad, CA), using the YAP/WWTR1 or species control IgG antibody for immunoprecipitation. Cross-links were reversed with heat and NaCl, then DNA isolated by phenol chloroform extraction. PCR primers were designed around the promoter of BDNF (Table 1). Physiology data were analyzed using Welch's t-test, when variances were not equal, and t-test, with P < 0.05 considered significant. All t-tests were performed as two-tailed, except where indicated in the text. Correlation analysis was performed by Pearson's correlation (using the stats package Hmisc on R) to identify parameters from physiological and expression data that correlate and cluster together. Expression data were analyzed initially using limma on R, with an interquartile range of 0.9, using the HTqPCR package.13Dvinge H. Bertone P. HTqPCR: high-throughput analysis and visualization of quantitative real-time PCR data in R.Bioinformatics. 2009; 25: 3325-3326Crossref PubMed Scopus (182) Google Scholar Correlation analysis of expression and physiology data were analyzed in both linear and logarithmic formats [−delta c(t)] to facilitate comparisons.13Dvinge H. Bertone P. HTqPCR: high-throughput analysis and visualization of quantitative real-time PCR data in R.Bioinformatics. 2009; 25: 3325-3326Crossref PubMed Scopus (182) Google Scholar Correlations with P < 0.05 and R2 > 0.40 were selected as potential candidates and plotted graphically. In addition, R, R2, and P values for correlations were plotted using R ggcorrplot packages to identify genes whose expression patterns were associated with physiological parameters. Post hoc Student's tests, Welch's t-tests, and analysis of variance of gene expression data were performed. For all tests, P values were considered significant if P < 0.05. PBO alone (without inhibitor) caused a significant increase in mean bladder mass (P < 0.005) and bladder/body mass ratio (P < 0.005) compared with sham animals (Figure 1, A and B). DAC-treated PBO animals showed further elevations in mean bladder mass (P < 0.05) and bladder/body mass ratio (P < 0.05, one-tailed t-test) versus PBO alone (Figure 1, A and B). Body mass at time of surgery varied from 250 to 300 g, and body mass was not significantly altered (Supplemental Figure S4, A–F and H). More important, DAC treatment had only minimal effects on final mass of sham bladders. Transnuclear cross-sectional SMC area was increased significantly during PBO, compared with sham (P < 0.01) (Figure 1, C and D). Consistent with bladder mass, the inc