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1.  Antiproliferative factor decreases Akt phosphorylation and alters gene expression via CKAP4 in T24 bladder carcinoma cells 
Background
Urinary bladder cancer is a common malignancy worldwide, and outcomes for patients with advanced bladder cancer remain poor. Antiproliferative factor (APF) is a potent glycopeptide inhibitor of epithelial cell proliferation that was discovered in the urine of patients with interstitial cystitis, a disorder with bladder epithelial thinning and ulceration. APF mediates its antiproliferative activity in primary normal bladder epithelial cells via cytoskeletal associated protein 4 (CKAP4). Because synthetic asialo-APF (as-APF) has also been shown to inhibit T24 bladder cancer cell proliferation at nanomolar concentrations in vitro, and because the peptide segment of APF is 100% homologous to part of frizzled 8, we determined whether CKAP4 mediates as-APF inhibition of proliferation and/or downstream Wnt/frizzled signaling events in T24 cells.
Methods
T24 cells were transfected with double-stranded siRNAs against CKAP4 and treated with synthetic as-APF or inactive control peptide; cells that did not undergo electroporation and cells transfected with non-target (scrambled) double-stranded siRNA served as negative controls. Cell proliferation was determined by 3H-thymidine incorporation. Expression of Akt, glycogen synthase kinase 3β (GSK3β), β-catenin, p53, and matrix metalloproteinase 2 (MMP2) mRNA was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Akt, GSK-3β, MMP2, β-catenin, and p53 protein expression, plus Akt, GSK-3β, and β-catenin phosphorylation, were determined by Western blot.
Results
T24 cell proliferation, MMP2 expression, Akt ser473 and thr308 phosphorylation, GSK3β tyr216 phosphorylation, and β-catenin ser45/thr41 phosphorylation were all decreased by APF, whereas p53 expression, and β-catenin ser33,37/thr41 phosphorylation, were increased by APF treatment in non-electroporated and non-target siRNA-transfected cells. Neither mRNA nor total protein expression of Akt, GSK3β, or β-catenin changed in response to APF in these cells. In addition, the changes in cell proliferation, MMP2/p53 mRNA and protein expression, and Akt/GSK3β/β-catenin phosphorylation in response to APF treatment were all specifically abrogated following CKAP4 siRNA knockdown.
Conclusions
Synthetic as-APF inhibits cell proliferation in T24 bladder carcinoma cells via the CKAP4 receptor. The mechanism for this inhibition involves regulating phosphorylation of specific cell signaling molecules (Akt, GSK3β, and β-catenin) plus mRNA and protein expression of p53 and MMP2.
doi:10.1186/1756-9966-29-160
PMCID: PMC3020166  PMID: 21143984
2.  A Synthetic Form of Frizzled 8-Associated Antiproliferative Factor Enhances p53 Stability through USP2a and MDM2 
PLoS ONE  2012;7(12):e50392.
Frizzled 8-associated Antiproliferative Factor (APF) is a sialoglycopeptide urinary biomarker of interstitial cystitis/painful bladder syndrome (IC/PBS), a chronic condition of unknown etiology with variable symptoms that generally include pelvic and/or perineal pain, urinary frequency, and urgency. We previously reported that native human APF suppresses the proliferation of normal bladder epithelial cells through a mechanism that involves increased levels of p53. The goal of this study was to delineate the regulatory mechanism whereby p53 expression is regulated by APF. Two APF-responsive cell lines (T24 bladder carcinoma cells and the immortalized human bladder epithelial cell line, TRT-HU1) were treated with asialo-APF (as-APF), a chemically synthesized form of APF. Biochemical analysis revealed that as-APF increased p53 levels in two ways: by decreasing ubiquitin specific protease 2a (USP2a) expression leading to enhanced ubiquitination of murine double minute 2 E3 ubiquitin ligase (MDM2), and by suppressing association of p53 with MDM2, thus impairing p53 ubiquitination. Biological responses to as-APF were suppressed by increased expression of wild type, but not mutant USP2a, which enhanced cell growth via upregulation of a cell cycle mediator, cyclin D1, at both transcription and protein levels. Consistent with this, gene silencing of USP2a with siRNA arrested cell proliferation. Our findings suggest that APF upregulates cellular p53 levels via functional attenuation of the USP2a-MDM2 pathway, resulting in p53 accumulation and growth arrest. These data also imply that targeting USP2a, MDM2, p53 and/or complex formation by these molecules may be relevant in the development of novel therapeutic approaches to IC/PBS.
doi:10.1371/journal.pone.0050392
PMCID: PMC3516501  PMID: 23236372
3.  Antiproliferative factor regulates connective tissue growth factor (CTGF/CCN2) expression in T24 bladder carcinoma cells 
Molecular Biology of the Cell  2012;23(10):1976-1985.
Connective tissue growth factor (CTGF/CNN2) is a novel APF target gene. A novel mechanism is described by which the APF cellular receptor, cytoskeleton-associated protein 4 (CKAP4), mediates APF-induced CTGF transcription.
Antiproliferative factor (APF) is a sialoglycopeptide elevated in the urine of patients with interstitial cystitis (IC)—a chronic, painful bladder disease of unknown etiology. APF inhibits the proliferation of normal bladder epithelial and T24 bladder carcinoma cells in vitro by binding to cytoskeleton-associated protein 4 (CKAP4) and altering the transcription of genes involved in proliferation, cellular adhesion, and tumorigenesis; however, specific molecular mechanisms and effector genes that control APF's antiproliferative effects are unknown. In this study, we found that there was a 7.5-fold up-regulation of connective tissue growth factor (CTGF/CCN2) expression in T24 bladder carcinoma cells treated with APF. Western blot revealed a dose-dependent increase in CCN2 protein levels, with secretion into the culture medium after APF treatment. CCN2 overexpression enhanced APF's antiproliferative activity, whereas CCN2 knockdown diminished APF-induced p53 expression. Using a luciferase reporter construct, we found that APF treatment resulted in fivefold activation of the CCN2 proximal promoter and, of importance, that small interfering RNA–mediated knockdown of CKAP4 inhibited CCN2 upregulation. In addition, we demonstrate that CKAP4 translocates to the nucleus and binds to the CCN2 proximal promoter in an APF-dependent manner, providing evidence that CCN2 regulation by APF involves CKAP4 nuclear translocation and binding to the CCN2 promoter.
doi:10.1091/mbc.E11-08-0714
PMCID: PMC3350560  PMID: 22438586
4.  Antiproliferative Factor Signaling and Interstitial Cystitis/Painful Bladder Syndrome 
A unique glycopeptide, antiproliferative factor (APF), has been suggested as a urinary biomarker and potential mediator of long-term bladder disorder Interstitial Cystitis/Painful Bladder Syndrome. There is no known cause for this disease. Several mechanistic approaches have been employed to address the underlying mechanism whereby APF regulates cellular responses in the bladder epithelium. A summary of recent literature is provided, and is focused on signal transduction pathways and networks that are responsive to APF.
doi:10.5213/inj.2011.15.4.184
PMCID: PMC3256302  PMID: 22259731
Human antiproliferative factor APF; Interstitial Cystitis; Signal transduction
5.  Interstitial cystitis antiproliferative factor (APF) as a cell-cycle modulator 
BMC Urology  2004;4:3.
Background
Interstitial cystitis (IC) is a chronic bladder disorder of unknown etiology. Antiproliferative factor (APF), a peptide found in the urine of IC patients, has previously been shown to decrease incorporation of thymidine by normal bladder epithelial cells. This study was performed to determine the effect of APF on the cell cycle of bladder epithelial cells so as to better understand its antiproliferative activity.
Methods
Explant cultures from normal bladder biopsy specimens were exposed to APF or mock control. DNA cytometry was performed using an automated image analysis system. Cell cycle phase fractions were calculated from the DNA frequency distributions and compared by two-way analysis of variance (ANOVA).
Results
APF exposure produced statistically significant increases in the proportion of tetraploid and hypertetraploid cells compared to mock control preparations, suggesting a G2 and/or M phase cell cycle block and the production of polyploidy.
Conclusions
APF has a specific effect on cell cycle distributions. The presence of a peptide with this activity may contribute to the pathogenesis of interstitial cystitis through disruption of normal urothelial proliferation and repair processes.
doi:10.1186/1471-2490-4-3
PMCID: PMC411044  PMID: 15068487
6.  p53 Mediates interstitial cystitis antiproliferative factor (APF)-induced growth inhibition of human urothelial cells 
FEBS letters  2007;581(20):3795-3799.
Antiproliferative factor (APF) is a sialoglycopeptide elevated in the urine of patients with interstitial cystitis, a urinary bladder disorder of unknown etiology that is characterized by chronic pelvic pain. The present study was directed toward uncovering a pathway through which APF signals. Treatment of human urothelial cells with native APF resulted in growth inhibition accompanied by blockade of cell cycle transit and increased p53. Reduced expression of p53 by RNA interference diminished, while ectopic expression of p53 mimicked, the effects of APF. These are the first findings implicating the network of p53 target genes in urothelial defects associated with interstitial cystitis.
doi:10.1016/j.febslet.2007.06.058
PMCID: PMC1939966  PMID: 17628545
Antiproliferative factor; Interstitial cystitis; Human urothelial cell; p53; p21Cip1/Waf1
7.  APF, HB-EGF, and EGF biomarkers in patients with ulcerative vs. non-ulcerative interstitial cystitis 
BMC Urology  2005;5:7.
Background
Interstitial cystitis (IC) is a chronic bladder disorder, with symptoms including pelvic and or perineal pain, urinary frequency, and urgency. The etiology of IC is unknown, but sensitive and specific biomarkers have been described, including antiproliferative factor (APF), heparin-binding epidermal growth factor-like growth factor (HB-EGF), and epidermal growth factor (EGF). However, the relative sensitivity of these biomarkers in ulcerative vs. nonulcerative IC is unknown, and these markers have yet to be validated in another laboratory. We therefore measured these markers in urine from patients with or without Hunner's ulcer, as well as normal controls, patients with bladder cancer, and patients with bacterial cystitis, at the First Hospital of China Medical University.
Methods
Urine specimens were collected from two groups of Chinese IC patients (38 IC patients with Hunner's ulcers, 26 IC patients without Hunner's ulcers), 30 normal controls, 10 bacterial cystitis patients and 10 bladder cancer patients. APF activity was determined by measuring 3H-thymidine incorporation in vitro, and HB-EGF and EGF levels were determined by ELISA.
Results
APF activity (inhibition of thymidine incorporation) was significantly greater in all IC patient urine specimens than in normal control specimens or in specimens from patients with bacterial cystitis or bladder cancer (p < 0.0001 for each comparison). Urine HB-EGF levels were also significantly lower and EGF levels significantly higher in both groups of IC patients than in the three control groups (p < 0.0001 for each comparison). Although APF and HB-EGF levels were similar in ulcerative and nonulcerative IC patients, EGF levels were significantly higher in IC patients with vs. without ulcers (p < 0.004).
Conclusion
These findings indicate that APF, HB-EGF and EGF are good biomarkers for both ulcerative and nonulcerative IC and validate their measurement as biomarkers for IC in Chinese patients.
doi:10.1186/1471-2490-5-7
PMCID: PMC1131910  PMID: 15862132
8.  Palmitoylation of Cytoskeleton Associated Protein 4 by DHHC2 Regulates Antiproliferative Factor-mediated Signaling 
Molecular Biology of the Cell  2009;20(5):1454-1463.
Previously, we identified cytoskeleton-associated protein 4 (CKAP4) as a major substrate of the palmitoyl acyltransferase, DHHC2, using a novel proteomic method called palmitoyl-cysteine identification, capture and analysis (PICA). CKAP4 is a reversibly palmitoylated and phosphorylated protein that links the ER to the cytoskeleton. It is also a high-affinity receptor for antiproliferative factor (APF), a small sialoglycopeptide secreted from bladder epithelial cells of patients with interstitial cystitis (IC). The role of DHHC2-mediated palmitoylation of CKAP4 in the antiproliferative response of HeLa and normal bladder epithelial cells to APF was investigated. Our data show that siRNA-mediated knockdown of DHHC2 and consequent suppression of CKAP4 palmitoylation inhibited the ability of APF to regulate cellular proliferation and blocked APF-induced changes in the expression of E-cadherin, vimentin, and ZO-1 (genes known to play a role in cellular proliferation and tumorigenesis). Immunocytochemistry revealed that CKAP4 palmitoylation by DHHC2 is required for its trafficking from the ER to the plasma membrane and for its nuclear localization. These data suggest an important role for DHHC2-mediated palmitoylation of CKAP4 in IC and in opposing cancer-related cellular behaviors and support the idea that DHHC2 is a tumor suppressor.
doi:10.1091/mbc.E08-08-0849
PMCID: PMC2649263  PMID: 19144824
9.  Intravesical Dimethyl Sulfoxide Inhibits Acute and Chronic Bladder Inflammation in Transgenic Experimental Autoimmune Cystitis Models 
New animal models are greatly needed in interstitial cystitis/painful bladder syndrome (IC/PBS) research. We recently developed a novel transgenic cystitis model (URO-OVA mice) that mimics certain key aspects of IC/PBS pathophysiology. This paper aimed to determine whether URO-OVA cystitis model was responsive to intravesical dimethyl sulfoxide (DMSO) and if so identify the mechanisms of DMSO action. URO-OVA mice developed acute cystitis upon adoptive transfer of OVA-specific OT-I splenocytes. Compared to PBS-treated bladders, the bladders treated with 50% DMSO exhibited markedly reduced bladder histopathology and expression of various inflammatory factor mRNAs. Intravesical DMSO treatment also effectively inhibited bladder inflammation in a spontaneous chronic cystitis model (URO-OVA/OT-I mice). Studies further revealed that DMSO could impair effector T cells in a dose-dependent manner in vitro. Taken together, our results suggest that intravesical DMSO improves the bladder histopathology of IC/PBS patients because of its ability to interfere with multiple inflammatory and bladder cell types.
doi:10.1155/2011/937061
PMCID: PMC2989383  PMID: 21113298
10.  Antiproliferative Factor-Induced Changes in Phosphorylation and Palmitoylation of Cytoskeleton-Associated Protein-4 Regulate Its Nuclear Translocation and DNA Binding 
Cytoskeleton-associated protein 4 (CKAP4) is a reversibly palmitoylated and phosphorylated transmembrane protein that functions as a high-affinity receptor for antiproliferative factor (APF)—a sialoglycopeptide secreted from bladder epithelial cells of patients with interstitial cystitis (IC). Palmitoylation of CKAP4 by the palmitoyl acyltransferase, DHHC2, is required for its cell surface localization and subsequent APF signal transduction; however, the mechanism for APF signal transduction by CKAP4 is unknown. In this paper, we demonstrate that APF treatment induces serine phosphorylation of residues S3, S17, and S19 of CKAP4 and nuclear translocation of CKAP4. Additionally, we demonstrate that CKAP4 binds gDNA in a phosphorylation-dependent manner in response to APF treatment, and that a phosphomimicking, constitutively nonpalmitoylated form of CKAP4 localizes to the nucleus, binds DNA, and mimics the inhibitory effects of APF on cellular proliferation. These results reveal a novel role for CKAP4 as a downstream effecter for APF signal transduction.
doi:10.1155/2012/150918
PMCID: PMC3320026  PMID: 22536245
11.  The Severity of Experimental Autoimmune Cystitis Can be Ameliorated by Anti-CXCL10 Ab Treatment 
PLoS ONE  2013;8(11):e79751.
Background
Interstitial cystitis (IC), more recently called painful bladder syndrome (PBS) is a complex disease associated with chronic bladder inflammation that primarily affects women. Its symptoms include frequent urinary urgency accompanied by discomfort or pain in the bladder and lower abdomen. In the United States, eight million people, mostly women, have IC/PBS. New evidence that autoimmune mechanisms are important in the pathogenesis of IC/PBS triggered interest.
Methodology/Principal Findings
SWXJ mice immunized with a homogenate of similar mice’s urinary bladders develop an autoimmune phenotype comparable to clinical IC with functional and histological alterations confined to the urinary bladder. Using the murine model of experimental autoimmune cystitis (EAC), we found that serum levels of CXCR3 ligand and local T helper type 1 (Th1) cytokine are elevated. Also, IFN-γ-inducible protein10 (CXCL10) blockade attenuated overall cystitis severity scores; reversed the development of IC; decreased local production of CXCR3 and its ligands, IFN-γ, and tumor necrosis factor-α (TNF-α); and lowered systemic levels of CXCR3 ligands. Urinary bladder CD4+ T cells, mast cells, and neutrophils infiltrates were reduced following anti-CXCL10 antibody (Ab) treatment of mice. Anti-CXCL10 Ab treatment also reversed the upregulated level of CXCR3 ligand mRNA at urinary bladder sites. The decreased number and percentage of systemic CD4+ T cells in EAC mice returned to normal after anti-CXCL10 Ab treatment.
Conclusion/Significance
Taken together, our findings provide important new information about the mechanisms underlying EAC pathogenesis, which has symptoms similar to those of IC/PBS. CXCL10 has the potential for use in developing new therapy for IC/PBS.
doi:10.1371/journal.pone.0079751
PMCID: PMC3836899  PMID: 24278169
12.  An hTERT-immortalized human urothelial cell line that responds to anti-proliferative factor 
Studies of the urothelium, the specialized epithelial lining of the urinary bladder, are critical for understanding diseases affecting the lower urinary tract, including interstitial cystitis, urinary tract infections and cancer. However, our understanding of urothelial pathophysiology has been hampered by a lack of appropriate model systems. Here, we describe the isolation and characterization of a non-transformed urothelial cell line (TRT-HU1), originally explanted from normal tissue and immortalized with hTERT, the catalytic subunit of telomerase. We demonstrate responsiveness of the cells to anti-proliferative factor (APF), a glycopeptide implicated in the pathogenesis of interstitial cystitis. TRT-HU1 carries a deletion on the short arm of chromosome 9, an early genetic lesion in development of bladder cancer. TRT-HU1 urothelial cells displayed growth and migration characteristics similar to the low-grade papilloma cell line RT4. In contrast, we observed marked differences in both phenotype and gene expression profiles between TRT-HU1 and the highly malignant T24 cell line. Together, these findings provide the first demonstration of a non-transformed, continuous urothelial cell line that responds to APF. This cell line will be valuable for studies of both benign and malignant urothelial cell biology.
doi:10.1007/s11626-010-9350-y
PMCID: PMC3029472  PMID: 21136194
TRT-HU1; Bladder; Urothelium; APF
13.  Prevention of Anti-microbial Peptide LL-37-Induced Apoptosis and ATP Release in the Urinary Bladder by a Modified Glycosaminoglycan 
PLoS ONE  2013;8(10):e77854.
Interstitial cystitis (IC), often referred to in combination with painful bladder syndrome, is a chronic inflammatory disease of the bladder. Current therapies primarily focus on replenishing urothelial glycosaminoglycan (GAG) layer using GAG analogs and managing pain with supportive therapies. However, the elusive etiology of IC and the lack of animal models to study the disease have been major hurdles developing more effective therapeutics. Previously, we showed an increased urinary concentration of antimicrobial peptide LL-37 in spina bifida patients and used LL-37 to develop a mouse model of cystitis that mimics important clinical findings of IC. Here we investigate (1) the molecular mechanism of LL-37 induced cystitis in cultured human urothelial cells and in mice, (2) the protective effects of GM-0111, a modified GAG, within the context of this mechanism, (3) the physiological and molecular markers that correlate with the severity of the inflammation, and (4) the protective effects of several GAGs using these biomarkers in our LL-37 induced cystitis model. We find that LL-37 quickly induces release of ATP and apoptosis in the urothelium. These changes can be inhibited by a chemically-modified GAG, GM-0111. Furthermore, we also find that GAG analogs provide varying degrees of protection against LL-37 challenge in mice. These findings suggest that GM-0111 and possibly GAG molecules prevent the development of cystitis by blocking the apoptosis and the concurrent release of ATP from the urothelium.
doi:10.1371/journal.pone.0077854
PMCID: PMC3813730  PMID: 24204996
14.  Bladder Pain Syndrome Treated with Triple Therapy with Gabapentin, Amitriptyline, and a Nonsteroidal Anti-Inflammatory Drug 
Purpose
Bladder pain syndrome is a chronic disease that manifests as bladder pain, frequency, nocturia, and urgency. Gabapentin, amitriptyline, and nonsteroidal anti-inflammatory drugs are efficacious treatments for bladder pain syndrome. Here, we assessed the effect of triple therapy with these drugs in women with bladder pain syndrome.
Methods
Between May 2007 and May 2010, we conducted a prospective nonrandomized study on 74 patients with bladder pain syndrome. Of these patients, 38 (11 men and 27 women; mean age, 55.9 years; range, 25 to 77 years; mean follow-up, 12.6 months) were administered the interstitial cystitis (IC) symptom scales (O'Leary-Sant Symptom Index) and visual analog scale (VAS) 1, 3, and 6 months after treatment to assess the efficacy of triple therapy.
Results
The pretreatment O'Leary-Sant IC symptom score was 11.7, and the post-treatment scores were 4.4, 3.8, and 4.0 at 1, 3, and 6 months, respectively; the pretreatment problem index score was 10.5, and the post-treatment scores were 3.7, 2.7, and 2.9 at 1, 3, and 6 months, respectively. The pretreatment VAS score was 6.7, and the post-treatment scores were 1.8, 1.5, and 1.7 at 1, 3, and 6 months, respectively. The O'Leary-Sant IC symptom index and problem index and VAS scores improved considerably 1 month after treatment (P<0.05). However, the results at 1, 3, and 6 months after treatment were not significantly different (P>0.05).
Conclusions
Triple therapy was sufficiently effective in patients with bladder pain syndrome and caused no significant adverse effects. However, large-scale studies should be performed to verify our findings.
doi:10.5213/inj.2010.14.4.256
PMCID: PMC3021818  PMID: 21253338
Bladder pain syndrome; Interstitial cystitis; Visual analog scale
15.  Structure–Activity Relationship Studies for the Peptide Portion of the Bladder Epithelial Cell Antiproliferative Factor from Interstitial Cystitis Patients 
Journal of medicinal chemistry  2008;51(19):5974-5983.
We performed comprehensive structure–activity relationship (SAR) studies on the peptide portion of antiproliferative factor (APF), a sialylated frizzled-8 related glycopeptide that inhibits normal bladder epithelial and urothelial carcinoma cell proliferation. Glycopeptide derivatives were synthesized by solid-phase methods using standard Fmoc chemistry and purified by RP-HPLC; all intermediate and final products were verified by HPLC-MS and NMR analyses. Antiproliferative activity of each derivative was determined by inhibition of 3H-thymidine incorporation in primary normal human bladder epithelial cells. Structural components of the peptide segment of APF that proved to be important for biological activity included the presence of at least eight of the nine N-terminal amino acids, a negative charge in the C-terminal amino acid, a free amino group at the N-terminus, maintenance of a specific amino acid sequence in the C-terminal tail, and trans conformation for the peptide bonds. These data provide critical guidelines for optimization of structure in design of APF analogues as potential therapeutic agents.
doi:10.1021/jm8002763
PMCID: PMC2778288  PMID: 18788730
16.  Mucosal Muscarinic Receptors Enhance Bladder Activity in Cats With Feline Interstitial Cystitis 
The Journal of urology  2009;181(3):1415-1422.
Purpose
Interstitial cystitis is a chronic pelvic pain syndrome of which the origin and mechanisms involved remain unclear. In this study Ca2+ transients in the bladder wall of domestic cats diagnosed with naturally occurring feline interstitial cystitis were examined.
Materials and Methods
Cross-sections of full-thickness bladder strips from normal cats and cats with feline interstitial cystitis were examined by optically mapping Ca2+ transients and recording tension. Responses of Ca2+ activity and detrusor contractions to pharmacological interventions were compared. In addition, pharmacological responses were compared in mucosa denuded preparations.
Results
Optical mapping showed that feline interstitial cystitis bladders had significantly more spontaneous Ca2+ transients in the mucosal layer than control bladders. Optical mapping also demonstrated that feline interstitial cystitis bladders were hypersensitive to a low dose (50 nM) of the muscarinic receptor agonist arecaidine when the mucosal layer was intact. This hypersensitivity was markedly decreased in mucosa denuded bladder strips.
Conclusions
In feline interstitial cystitis cat bladders there is increased Ca2+ activity and sensitivity of muscarinic receptors in the mucosal layer, which can enhance smooth muscle spontaneous contractions.
doi:10.1016/j.juro.2008.10.138
PMCID: PMC3686310  PMID: 19157447
urinary bladder; cystitis, interstitial; cats; cat diseases; receptors, muscarinic
17.  Herpes simplex virus vector-mediated gene delivery for the treatment of lower urinary tract pain 
Gene therapy  2009;16(4):558-569.
Interstitial cystitis (IC)/painful bladder syndrome (PBS) is a painful debilitating chronic visceral pain disorder of unknown etiology that affects an estimated 1 million people in the, United States alone. It is characterized by inflammation of the bladder that results in chronic pelvic pain associated with bladder symptoms of urinary frequency and urgency. Regardless of the etiology, IC/PBS involves either increased and/or abnormal activity in afferent nociceptive sensory neurons. Pain-related symptoms in patients with IC/PBS are often very difficult to treat. Both medical and surgical therapies have had limited clinical utility in this debilitating disease and numerous drug treatments, such as heparin, dimethylsulfoxide and amitriptyline, have proven to be palliative at best, and in some IC/PBS patients provide no relief whatsoever. Although opiate narcotics have been employed to help alleviate IC/PBS pain, this strategy is fraught with problems as systemic narcotic administration causes multiple unwanted side effects including mental status change and constipation. Moreover, chronic systemic narcotic use leads to dependency and need for dose escalation due to tolerance: therefore, new therapies are desperately needed to treat refractory IC/PBS. This has led our group to develop a gene therapy strategy that could potentially alleviate chronic pelvic pain using the herpes simplex virus-directed delivery of analgesic proteins to the bladder.
doi:10.1038/gt.2009.19
PMCID: PMC2732574  PMID: 19242523
interstitial cystitis; painful bladder syndrome; visceral pain; dorsal root ganglia; herpes simplex virus
18.  Development, Validation and Testing of an Epidemiological Case Definition of Interstitial Cystitis/Painful Bladder Syndrome 
The Journal of urology  2010;183(5):1848-1852.
Purpose
No standard case definition exists for interstitial cystitis/painful bladder syndrome for patient screening or epidemiological studies. As part of the RAND Interstitial Cystitis Epidemiology study, we developed a case definition for interstitial cystitis/painful bladder syndrome with known sensitivity and specificity. We compared this definition with others used in interstitial cystitis/painful bladder syndrome epidemiological studies.
Materials and Methods
We reviewed the literature and performed a structured, expert panel process to arrive at an interstitial cystitis/painful bladder syndrome case definition. We developed a questionnaire to assess interstitial cystitis/painful bladder syndrome symptoms using this case definition and others used in the literature. We administered the questionnaire to 599 women with interstitial cystitis/painful bladder syndrome, overactive bladder, endometriosis or vulvodynia. The sensitivity and specificity of each definition was calculated using physician assigned diagnoses as the reference standard.
Results
No single epidemiological definition had high sensitivity and high specificity. Thus, 2 definitions were developed. One had high sensitivity (81%) and low specificity (54%), and the other had the converse (48% sensitivity and 83% specificity). These values were comparable or superior to those of other epidemiological definitions used in interstitial cystitis/painful bladder syndrome prevalence studies.
Conclusions
No single case definition of interstitial cystitis/painful bladder syndrome provides high sensitivity and high specificity to identify the condition. For prevalence studies of interstitial cystitis/painful bladder syndrome the best approach may be to use 2 definitions that would yield a prevalence range. The RAND Interstitial Cystitis Epidemiology interstitial cystitis/painful bladder syndrome case definitions, developed through structured consensus and validation, can be used for this purpose.
doi:10.1016/j.juro.2009.12.103
PMCID: PMC3519367  PMID: 20303099
urinary bladder; cystitis, interstitial; pain; epidemiology; diagnosis
19.  Alterations in the non-neuronal acetylcholine synthesis and release machinery in esophageal epithelium 
Life sciences  2012;91(21-22):1065-1069.
Aims
A non-neuronal cholinergic system has been described in epithelial cells including that of the urinary bladder (urothelium) and the upper gastrointestinal tract (esophagus). Epithelial dysfunction has been implicated in the pathophysiology of persistent pain conditions such as painful bladder syndrome as well as functional heartburn. For example, alterations in the ability to synthesize and release acetylcholine may contribute to changes in epithelial sensory and barrier function associated with a number of functional genitourinary and intestinal disorders.
Main methods
We examined using immunoblot, acetylcholine (ACh)-synthesis and release components in cat esophageal mucosa and whether elements of these components are altered in a naturally occurring model of chronic idiopathic cystitis termed feline interstitial cystitis (FIC).
Key findings
We identified proteins involved in ACh synthesis and release (high affinity choline transporter, CHT1; ACh synthesizing enzymes choline acetyltransferase ChAT and carnitine acetyltransferase CarAT; vesicular ACh transporter VAChT and the organic cation transporter isoforms 1-3 or OCT1-3) in cat esophageal mucosa. Significant alterations in CHT, ChAT, VAChT and OCT-1 were detected in the esophageal mucosa from FIC cats. Changes in the vesicular nucleotide transporter (VNUT) and the junctional protein pan-cadherin were also noted.
Significance
Taken together, these findings suggest that changes in the non-neuronal cholinergic system may contribute to alterations in cell-cell contacts and possibly communication with underlying cells that may contribute to changes in sensory function and visceral hyperalgesia in functional esophageal pain.
doi:10.1016/j.lfs.2012.04.028
PMCID: PMC3435437  PMID: 22569297
esophageal mucosa; barrier function; signaling function
20.  Crucial Role of TRPC1 and TRPC4 in Cystitis-Induced Neuronal Sprouting and Bladder Overactivity 
PLoS ONE  2013;8(7):e69550.
Purpose
During cystitis, increased innervation of the bladder by sensory nerves may contribute to bladder overactivity and pain. The mechanisms whereby cystitis leads to hyperinnervation of the bladder are, however, poorly understood. Since TRP channels have been implicated in the guidance of growth cones and survival of neurons, we investigated their involvement in the increases in bladder innervation and bladder activity in rodent models of cystitis.
Materials and Methods
To induce bladder hyperactivity, we chronically injected cyclophosphamide in rats and mice. All experiments were performed a week later. We used quantitative transcriptional analysis and immunohistochemistry to determine TRP channel expression on retrolabelled bladder sensory neurons. To assess bladder function and referred hyperalgesia, urodynamic analysis, detrusor strip contractility and Von Frey filament experiments were done in wild type and knock-out mice.
Results
Repeated cyclophosphamide injections induce a specific increase in the expression of TRPC1 and TRPC4 in bladder-innervating sensory neurons and the sprouting of sensory fibers in the bladder mucosa. Interestingly, cyclophosphamide-treated Trpc1/c4−/− mice no longer exhibited increased bladder innervations, and, concomitantly, the development of bladder overactivity was diminished in these mice. We did not observe a difference neither in bladder contraction features of double knock-out animals nor in cyclophosphamide-induced referred pain behavior.
Conclusions
Collectively, our data suggest that TRPC1 and TRPC4 are involved in the sprouting of sensory neurons following bladder cystitis, which leads to overactive bladder disease.
doi:10.1371/journal.pone.0069550
PMCID: PMC3726658  PMID: 23922735
21.  Cystitis, Co-morbid Disorders and Associated Epithelial Dysfunction 
Neurourology and urodynamics  2011;30(5):668-672.
Introduction
Interstitial cystitis (painful bladder syndrome / interstitial cystitis; PBS/IC) is a persistent pain syndrome affecting the urinary bladder with symptoms including urinary frequency, bladder pain and nocturia.(1–6) Various animal models have been studied, most of which mimic some aspect of the human condition of interest to the investigator(s). This review will provide examples of various animal models including those incorporating chronic stress, thought to produce features that share similarities to that of PBS/IC patients, whose symptoms are often exacerbated by various stressors. (7–12)
This review also provides evidence that patients with PBS/IC exhibit abnormalities within the bladder epithelium (or urothelium), even though a consistent relationship of such changes with symptom severity has not been demonstrated. These changes include alterations in urothelial integrity, differentiation and/or proliferation as well as changes in ‘sensory’ function (altered expression or sensitivity of receptors and ion channels).
Establishing a diagnostic ‘indicator’ with a high degree of correlation in this syndrome would be of value in terms of disease status, diagnosis and treatment. There have been reports of a number of factors/mediators altered in PBS/IC. However, the lack of a validated biomarker and a well-defined etiology for this syndrome introduces a number of complications, including diagnostic confidence, choice of appropriate animal models to study basic mechanism with the goal toward treatment, and rational therapies.
It is also becoming increasingly apparent that patients with PBS/IC often overlap or share symptoms commonly associated with other persistent pain disorders. These include (but are not limited to) irritable bowel syndrome (IBS), non-cardiac chest pain, fibromyalgia and even overactive bladder syndrome (OAB).(13–18) Such types of changes are not limited to the urinary bladder, however, as reports of alterations in epithelial signaling/barrier function have been described in patients diagnosed with a wider variety of syndromes, including functional and inflammatory bowel disorders such as irritable bowel syndrome (IBS), gastrointestinal esophageal reflux disease (GERD) and asthma.(19–21) These and other findings suggest that changes within the epithelium (barrier as well as signaling functions) may be a common occurrence that may contribute to peripheral mechanisms of hypersensitivity in a number of disorders.
doi:10.1002/nau.21109
PMCID: PMC3113628  PMID: 21661011
bladder urothelium; esophageal epithelium; sensor function; hypersensitivity
22.  Changes in ATP Stimulated ATP Release Suggests an Association between Cytokine and Purinergic Signaling in Bladder Urothelial Cells 
Urology  2009;74(5):1163-1168.
Objective
To determine whether antiproliferative factor (APF) or epidermal growth factor (EGF) can induce changes in purinergic signaling in normal bladder urothelial cells (BUC) and/or whether antagonizing EGF activity or blocking ATP-purinergic receptors can induce changes in purinergic signaling in interstitial cystitis (IC) cells.
Methods
IC and normal BUC were obtained from patients’ bladder biopsies. IC BUC were treated with genistein, which antagonizes EGF’s activity, while normal BUC were treated with EGF, mock APF, or APF. Suramin, which antagonizes ATP activity, was used to treat APF-treated normal BUC. ATP release was determined by stimulating BUC with 30μM ATP and then collecting supernatant over a 3-hour period. ATP quantification was measured by luciferin-luciferase assay. P2X3 expression on BUC was determined by fluorescence activated cell sorting (FACS).
Results
Genistein treatment of IC BUC resulted in significantly decreased ATP release, thus reverting IC cells to a normal purinergic signaling phenotype. Conversely, normal BUC treated with EGF or APF resulted in significantly increased ATP release and P2X3 expression, converting normal BUC to an IC phenotype. Suramin treatment of APF-treated normal BUC significantly reduced ATP release.
Conclusions
Genistein and suramin reversed the augmented ATP release in IC BUC and APF-treated normal BUC respectively, suggesting the possibility of intravesical use of these agents in IC treatment. EGF and APF induced augmented purinergic signaling in normal BUC as determined by increased ATP release and increased P2X3 expression. These data suggest an association between cytokines and purinergic signaling in human BUC that should be explored further.
doi:10.1016/j.urology.2009.02.066
PMCID: PMC2777753  PMID: 19628257
anti-proliferative factor (APF); adenosine triphosphate (ATP); Bladder urothelial cells (BUC); epidermal growth factor (EGF); heparin-binding epidermal growth factor-like growth factor (HB-EGF); interstitial cystitis (IC)
23.  Proteomic Investigation on Chronic Bladder Irritation in the Rat 
Urology  2008;71(3):536-540.
Purpose
Interstitial cystitis (IC) is a painful bladder syndrome associated with urinary frequency and urgency. Elusive etiology of IC makes its diagnosis only possible by exclusion in many cases. In the present study, we used proteomics for identifying disease-associated proteins in rat model of chronic bladder irritation.
Materials & Methods
Chronic irritation of rat bladder was caused by a brief (90 secs) intravesical instillation of 0.2ml of 0.4N HCl. Whole bladders were collected at different time points after treatment, snap frozen and nuclear and cytosolic protein extracts were obtained. Samples were resolved in standard 2D-gels stained with an improved Coomasie Stain or by Differential Gel Electrophoresis (DIGE). Differentially expressed spots were excised and identified by MALDI-TOF MS/MS. Histological and Western blot analysis were also performed.
Results
Bladder morphology and histologic appearance of bladder sections following HCl treatment reflected hemorrhage, edema, epithelial denudation, detrusor mastocytosis and eosinophilia. Proteomic analysis of irritated rat bladder revealed marked overexpression of four nuclear proteins and marked underexpression of one nuclear protein compared to normal rat bladders. Among these proteins, inflammation-associated Calgranulin A (over) and smooth muscle protein-22/transgelin (under) showed opposed expression patterns following bladder irritation.
Conclusions
Presence of mast cells and eosinophils and overexpression of calgranulin A confirm the inflammatory component of HCl-irritated bladder. Altered expression of nuclear proteins is of particular interest because of their possible role as a prognostic marker in inflammatory bladder disorders. However, more studies are needed before clinical application of these findings can be established.
doi:10.1016/j.urology.2007.10.069
PMCID: PMC3750729  PMID: 18342204
Interstitial Cystitis; Proteomics; Biomarkers; 2-D Gel
24.  NAD(P)H:Quinone oxidoreductase 1 (NQO1) protects bladder epithelium against painful bladder syndrome in mice 
Free radical biology & medicine  2012;53(10):1886-1893.
Painful Bladder Syndrome (PBS) or interstitial cystitis (IC) is a poorly understood chronic disease, which is characterized by thinning of bladder epithelium and intense pain. Here we demonstrate that NAD(P)H:quinone oxidoreductase 1−/− (NQO1−/−) mice developed in our laboratory represent a new animal model of PBS. NQO1 is known to protect against physiological stress as well as transcription factors against proteasomal degradation. In the present study we demonstrate that NQO1 is necessary for bladder epithelium integrity, and to prevent the development/progression of PBS. We observed down regulation of energy metabolism, adhesion, and apoptotic signaling cascades, which led to mitochondrial aberrations and profound alterations in energy metabolism, increased susceptibility to ROS generation and apoptosis in luminal epithelium in NQO1−/− mice that were absent in wild-type mice. These pathophysiological changes led to incidence of PBS in NQO1−/− mice. The results together demonstrate for the first time that NQO1 is an endogenous factor in protection against PBS.
doi:10.1016/j.freeradbiomed.2012.08.584
PMCID: PMC3495563  PMID: 22985937
NQO1; bladder; Interstitial Cystitis; Painful Bladder Syndrome
25.  RDP58 inhibits T cell-mediated bladder inflammation in an autoimmune cystitis model 
Journal of autoimmunity  2007;30(4):257-265.
Interstitial cystitis (IC) is a chronic inflammatory condition of the urinary bladder with a strong autoimmune component. Currently, the major challenge in IC treatment is the development of effective therapies. RDP58 is a novel D-amino acid decapeptide with potent immunosuppressive activity. In this study, we investigated whether RDP58 was effective as an intravesical agent for treating bladder autoimmune inflammation in a transgenic mouse model (URO-OVA mice). URO-OVA mice were adoptively transferred with syngeneic activated splenocytes of OT-I mice transgenic for the OVA-specific CD8+ TCR for cystitis induction and treated intravesically with RDP58 at days 0 and 3. Compared with controls, the RDP58-treated bladders showed markedly reduced histopathology and expressions of mRNAs and proteins of TNF-α, NGF and substance P. To determine whether the inhibition of bladder inflammation by RDP58 was due to the interference with effector T cells, we treated the cells with RDP58 in vitro. Cells treated with RDP58 showed reduced production of TNF-α and IFN-γ as well as apoptotic death. Collectively, these results indicate that RDP58 is effective on treating T cell-mediated experimental autoimmune cystitis and may serve as a useful intravesical agent for the treatment of autoimmune-associated bladder inflammation such as IC.
doi:10.1016/j.jaut.2007.10.005
PMCID: PMC2441447  PMID: 18162370
Autoimmunity; Cystitis; Inflammation; RDP58; Urinary Bladder

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