Role of the vascular endohelial growth factor in bladder pain syndrome/interstitial cystitis

ROLE OF VASCULAR ENDOTHELIAL GROWTH FACTOR IN BLADDER PAIN SYNDROME/ INTERSTITIAL CYSTITIS ANA JÚLIA DOS REIS PEREIRA Dissertação do Mestrado em Bioquímica Universidade do Porto Faculdade de Ciências Instituto de Ciências Biomédicas Abel Salazar 2012 ANA JÚLIA DOS REIS PEREIRA ROLE OF VASCULAR ENDOTHELIAL GROWTH FACTOR IN BLADDER PAIN SYNDROME/ INTERSTITIAL CYSTITIS Dissertação de Candidatura ao grau de Mestre em Bioquímica da Universidade do Porto Orientador(a): Doutora Ana Cristina Estrela de Oliveira Charrua Cordeiro Categoria: Bolseira de Pós-Doutoramento (Investigadora) Afiliação: Faculdade de Medicina da Universidade do Porto Co-orientador(a): Professora Doutora Célia da Conceição Duarte Cruz Categoria: Professora Auxiliar Afiliação: Faculdade de Medicina da Universidade do Porto 2012 Table of Contents Table of Contents Table of Contents ............................................................................................................. 1 List of Abbreviations and Symbols .................................................................................... 2 Abstract ............................................................................................................................ 5 Resumo ............................................................................................................................ 7 Introduction ....................................................................................................................... 9 Bladder Pain Syndrome/Interstitial Cystitis (BPS/IC) ....................................................10 VEGF family and their receptors...................................................................................11 VEGF molecules and their receptors in BPS/IC ............................................................13 Materials and Methods .....................................................................................................15 Animals ........................................................................................................................16 Animal model ...............................................................................................................16 Immunohistochemistry and Image acquisition ..............................................................16 Enzyme-linked immunosorbent assay (ELISA) .............................................................17 Creatinine Assay ..........................................................................................................18 Statistical Analysis........................................................................................................18 Results ............................................................................................................................19 Expression of VEGF in the urothelium ..........................................................................20 Expression of PlGF in the urothelium ...........................................................................21 Expression of VEGFR1 in the urothelium .....................................................................22 Expression of VEGF in the detrusor muscle .................................................................23 Expression of VEGFR1 in the detrusor muscle .............................................................24 VEGF quantification in urine .........................................................................................25 Discussion .......................................................................................................................26 VEGF, PlGF and VEGFR1 expression upon chronic adrenergic stimulation in the urothelium ....................................................................................................................27 VEGF, PlGF and VEGFR1 expression upon chronic adrenergic stimulation in detrusor smooth muscle .............................................................................................................28 VEGF quantification in urine .........................................................................................29 Conclusions .....................................................................................................................30 Future Prospects .............................................................................................................32 Acknowledgements ..........................................................................................................34 Bibliography .....................................................................................................................37 2 List of Abbreviations and Symbols 3 List of Abbreviations and Symbols α1adrenergic – Alpha1-Adrenergic Akt/PKB – Protein Kinase B Bax – Bcl-2-associated X protein BPS/IC – Bladder Pain Syndrome/ Interstitial Cystitis Ca2+ – Calcium cation 2+ Caspase 3 – Cysteine-Aspartic-Acid-Protease Member 3 DAB – diaminobenzidine tetrahydrochloride hydrate ELISA – Enzyme-Linked Immunosorbent Assay PHE – Phenylephrine NGF – Nerve Growth Factor NO – Nitric Oxide NRP(s) – Neuropilin(s) NRP2 – Neuropilin 2 PBS – Phosphate Buffer Saline PBST – Phosphate Buffer Saline + Triton X-100 PKC – Protein Kinase C PLCγ – Phospholipase C gamma PlGF – Placental Growth Factor PlGF-IR – Placental Growth Factor Immunoreaction VEGF – Vascular Endothelial Growth Factor VEGFA – Vascular Endothelial Growth Factor Member A VEGFA120 – Vascular Endothelial Growth Factor Member A Isoform 120 VEGFA121 – Vascular Endothelial Growth Factor Member A Isoform 121 VEGFA145 – Vascular Endothelial Growth Factor Member A Isoform 145 VEGFA165 – Vascular Endothelial Growth Factor Member A Isoform 165 VEGFA189 – Vascular Endothelial Growth Factor Member A Isoform 189 VEGFA206 – Vascular Endothelial Growth Factor Member A Isoform 206 VEGFB – Vascular Endothelial Growth Factor Member B VEGFC – Vascular Endothelial Growth Factor Member C VEGFD – Vascular Endothelial Growth Factor Member D VEGF-IR – Vascular Endothelial Growth Factor Immunoreaction VEGFR1 – Vascular Endothelial Growth Factor Receptor 1 VEGFR1-IR – Vascular Endothelial Growth Factor Immunoreaction VEGFR2 – Vascular Endothelial Growth Factor Receptor 2 4 VEGFR3 – Vascular Endothelial Growth Factor Receptor 3 VEGFRs – Vascular Endothelial Growth Factor Receptors TRPV1 – Transient Receptor Potential Cation Channel 5 Abstract 6 Abstract Bladder pain syndrome/Interstitial Cystitis (BPS/IC) is characterized by suprapubic pain related to bladder filling, usually accompanied by urgency and nocturia, in the absence of urinary infection or other pathology. Recently, it has been proposed a hypothesis that hyperactivity of the sympathetic nervous system may be associated with the altered pain sensation, bladder histological changes and/or urinary frequency associated with BPS/IC. Vascular Endothelial Growth Factor family molecules and its receptors are overexpressed in the bladder wall of BPS/IC patients and in animal models. In this study, it is hypothesized that sympathetic dysfunction, induced by chronic administration of phenylephrine (PHE), may result in changes in VEGF expression and function in the urinary bladder. The expression of Vascular Endothelial Growth Factor (VEGF), Placental Growth Factor (PlGF) and Vascular Endothelial Growth Factor Receptor 1 (VEGFR1) in the urinary bladder (urothelium and detrusor muscle cells) was analysed by immunohistochemistry. It was observed basal expression of VEGF and PlGF in the urinary bladder (urothelium and detrusor muscle cells) from control animals. In PHEtreated rats, an increase of VEGF, PlGF and VEGFR1 expression was observed in areas of damaged urothelium, characterized by low urothelium. VEGF expression and VEGFR1 in the detrusor muscle from treated rats was not altered, when compared to their expression in control animals. VEGF urinary levels were also determined by ELISA. The results obtained for concentration of VEGF in urine from PHE-treated rats and the one in urine from control rats were inconclusive. We can speculate that VEGF and related molecules may have a relevant contribution to inflammatory and apoptotic events in chronic adrenergic stimulated animals. 13 . Figure 2: Localization of VEGF receptors 1 and 2 homodimeric and heterodimeric structures and respective ligands (Olsson et al., 2006) VEGFA and its splice variants activity vary according to their different abilities to interact with VEGFR co-receptors (neuropilins and heparan sulphate proteoglycans) (Lee, et al., 2005; Woolard et al., 2004). In human bladder urothelium, mounting evidence suggests a clear abnormality in the distribution of VEGF-Rs and neuropilins (NRP), which might be important in pathophysiology and diagnosis of BPS/IC (Saban et al., 2008a). VEGF molecules and their receptors in BPS/IC VEGF expression is related to the modifications that might occur in the bladder vascular system (Saban et al., 2011). VEGF has a strong expression in the urothelium, as well as a slightest expression in endothelial cells. In lamina propria, VEGF expression is mild (Kiuchi et al., 2009). VEGF receptors are functionally active and localised in urothelial and neuronal cells in the urinary bladder, suggesting potential physiological functions of VEGF signalling in these cells (Saban et al., 2008b). Human umbrella cells have an intense expression of VEGFR1 and NRP2, contrasting with their poor expression in the lamina propria and the detrusor muscle (Saban et al., 2008a). In these last two layers, they seem confined both to blood vessels and inflammatory cells (Saban et al., 2008a). It has been hypothesized that the VEGF receptors expressed in non-endothelial cells are involved in mechanism of cell survival (Saban et al., 2008b). In BPS/IC some of the above mentioned proteins expression changes drastically. VEGF levels are increased in lamina propria and diffuse in the extracellular space of 14 BPS/IC patients that present glomerulations under hydrodistension (Kiuchi et al., 2009; Saban et al., 2011; Saban et al., 2008a). In addition, experiments using animal models of urinary bladder inflammation demonstrated that VEGF expression increases in the bladder wall (Saban et al., 2011). Increased expression of VEGF during cystitis is involved in nerve plasticity (Saban et al., 2011). In fact, VEGF induces an increase in substance P and TRPV1 expression, two molecules known to be expressed in urinary bladder nociceptors (Saban et al., 2011), indicating an additional role in the control of pain pathway. PlGF expression and role in BPS/IC is unknown. VEGFR1 and NRP2 expression is decreased during BPS/IC (Saban et al., 2008a). Since VEGF molecules and their receptors have a role in cell survival in normal conditions, it has been hypothesized that the observed changes in the expression of these molecules represent an additional layer of protection for urothelium (Saban et al., 2008a). However, if in the urothelium they present a similar role to the one observed in the control of vascular permeability and angiogenesis, they can be involved in urothelial leakage observed in BPS/IC (Saban et al., 2008a). Recent observations seem to indicate that there is a linkage between sympathetic dysfunction and VEGF molecules and their receptor activity. Overactivity of sympathetic nervous system may lead to degeneration of the bladder tissue, loss of bladder cells, angiogenesis and inflammation (Birder et al., 2011; Gamper et al., 2009; Kiuchi et al., 2009). Increased sympathetic activity results in an increased noradrenaline release (Stein et al., 1999) which has apoptotic effects on urothelial cells, promote bladder inflammation and sensitize bladder nociceptors leading to bladder hyperactivity and increased noxious input (Charrua et al., 2011). In fact, increased sympathetic activity, neuron density, and vasomotor tone have been demonstrated in bladder tissues from patients suffering from BPS/IC (Stein et al., 1999). The main goal of the present work is to understand if there is a relation between sympathetic dysfunction and VEGF molecules and their receptors expression. In order to achieve this goal the bladder of chronic adrenergic stimulated rats was analysed for the expression of VEGF, PlGF and VEGFR1, by performing immunohistochemistry for these proteins and Enzyme-linked Immunoenzymatic Assay (ELISA). 15 Materials and Methods 16 Materials and Methods Animals Twelve female Wistar rats (Charles River Laboratories, Barcelona, Spain) with an average weight of 275g were used. Animals were maintained in the animal house at 22 ºC and 60% humidity under a 12-hour light-dark cycle. All experiments were performed according to the European Communities Council Directive of 24 November 1986 (86/609/EEC). Animal model Animals were divided in two groups of six animals each: control group and treated group. Animals from control group received subcutaneous injection of saline solution and treated group received subcutaneous injection of 2.5 mg/Kg phenylephrine, both daily for 14 days. At day 15, animals were anaesthetized with sodium pentobarbital 20% and the urine was collected to two tubes, one for each group of animals, and stored at -20ºC until further use. The urinary bladders were harvested and fixed in 10% formalin, overnight at 4˚C. Then they were dehydrated, included in paraffin and sectioned at 5 µm. Immunohistochemistry and Image acquisition Bladder sections were deparaffinised and rehydrated (xylol, ethanol 100%, ethanol 90%, ethanol 75% and distilled water, 5 minutes each). Antigen retrieval was achieved by heating the bladder slides immersed in 3g/L citrate buffer pH=6.0, in microwave oven, in maximum potency until boiling (around 7 minutes), then boiled for one minute, and then up to 15 minutes in the lowest potency. Slides were let in citrate buffer at room temperature until complete cooling. Then they were washed in 0.1 M Phosphate Buffer Saline (PBS) (10 minutes), and endogenous peroxidase was blocked in 1% hydroxide peroxide in methanol (v/v), for 10 minutes. After washing once more with 0.1 M PBS (10 minutes), the tissue was blocked with a solution of 10% normal horse serum in 0.1 M Phosphate Buffer Saline + Triton X-100 (PBST). Three sections of each animal were incubate with each of the following primary antibody, at 4ºC, two overnights: 1:50 rabbit anti-VEGF (sc-152 (A-20) Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), 1:100 goat anti-PlGF (sc-27135 (K-20), Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), 17 1:50 rabbit anti-VEGFR1 (RB-1527-P, Neomarkers, Fremont, CA). After three washes with 2% normal swine serum in PBST, for 10 minutes each, tissues were incubated with a 1:200 swine anti-rabbit secondary antibody (Dako, Glostrup, Denmark) and 1:200 HRP conjugate donkey anti-goat (catalogue no. sc-2020, Santa Cruz Biotechnology Inc.), for 1 hour at room temperature. The secondary antibody was washed twice with 2% normal swine serum in PBST, for 10 minutes, and once with PBST, for 10 minutes. Tissues were then treated with 1:200 horseradish peroxidase combined with biotin and avidin (VECTASTAIN® Elite ABC Reagent), for 1 hour, at room temperature. After washing with 0.1 M PBS (5 minutes) and with 0.1 M Tris-HCl (5 minutes), the tissue was incubated with 1.4 mM 3,3’-diaminobenzidine tetrahydrochloride hydrate (DAB) + 1: 4000 hydrogen peroxide in a 0.05 M Tris-HCl solution. DAB solution was washed with 0.1 M Tris-HCl (5 minutes), 0.1 M PBS (5 minutes) and distilled water (5 minutes), and then counterstained with Hematoxylin. Bladder sections were then dehydrated (distilled water, ethanol 70%, ethanol 90%, ethanol 100%, xylol, each for 5 minutes) and mounted using Entellan Neu. All tissues were visualized with a Zeiss Vision Axioskop 40 photomicroscope and imaged using a AxioCam MRc5 camera driven by Axion Vision Rel 4.8 software. Immunopositive staining was evaluated by measuring the optical density on an arbitrary scale between 0— white pixels and 255—black pixels in three urothelial or detrusor areas per bladder section with National Institutes of Health ImageJ 1.43u software. The average optical density in each section was then calculated. Enzyme-linked immunosorbent assay (ELISA) Enzyme-linked immunosorbent assay (ELISA) kit was used to quantify urinary VEGF. Urine from control (n=6) and PHE-treated rats (n=6) were assayed as described below, using RayBio® Rat VEGF ELISA Kit. Briefly, 96-well plates were coated with antiVEGF polyclonal antibodies. Urine or VEGF standards were added to each well, and plates were incubated. After several washes, anti-VEGF monoclonal antibodies were added, and plates were again incubated. After thorough washes, the amount of bound antibody was detected using immunoglobulin horseradish peroxidase-streptavidin conjugate antibody. The unbound conjugate was washed out, followed by 30 minute incubation with tetramethylbenzidine, at room temperature. Stop solution was added to terminate the reactions. Colour change was measured with a microplate reader TECAN infinite® 200 No. 30017581 (Mannëdorf, Switzerland) at 450 nm. All samples were run to duplicate, and values were averaged. VEGF levels were normalized to the urinary concentration of creatinine. 18 Creatinine Assay The same two pools of urine used for VEGF quantification were assayed for creatinine quantification, according to the specified protocol and using Creatinine (urinary) Assay Kit (Item No. 500701, Cayman Chemical Company, USA). Sample or Creatinine standard were added to the respective wells. After incubation with alkaline picrate for 10 min, the absorbance was read at 495 nm (initial absorbance). After 20 min of incubation with acid solution, absorbance was read at 495 nm (final absorbance). The difference between the initial and the final absorbance reading of each standard, followed by the correction of the values subtracting the background value, generated the adjusted creatinine standard curve. The sample creatinine concentration was determined using the adjusted creatinine standard curve, previously subtracting initial and final absorbance readings and, to the total of this difference, subtracting the background value. Statistical Analysis Results are presented as the mean ± standard deviation. Mean differences were compared by one-way ANOVA, followed by the post hoc Bonferroni test for multiple comparisons using SigmaStat® 2.03 software with p<0.001 considered statistically significant. 19 Results 20 Results Expression of VEGF in the urothelium In the current work, we aimed at observing the expression of VEGF in the urothelium from control and PHE-treated rats. This animal model was employed to characterize the influence of chronic adrenergic stimulation in the urinary bladder, more specifically the urothelium, considering the expression of VEGF and related molecules. An initial assessment of VEGF levels was performed using immunohistochemistry. Intensity signal of VEGF immunoreaction (VEGF-IR) in the urothelium of the urinary bladder of control animals was 109 +/- 47 (Figure 3A and 3D). In PHE-treated animals, the signal intensity of VEGF-IR was 86 +/- 61 in normal urothelium (Figure 3B and 3D) and 311 +/- 180 in regions with low urothelium (Figure 3C and 3D). Figure 3: Photomicrographs of VEGF expression in the urothelium of control rats (A) and in high (B) and low urothelium (C) from PHE 2.5 mg/Kg -treated rats. D: Mean signal intensity of VEGF expression in the urothelium of a control and in the normal urothelium areas (high uro) and low urothelium areas (low uro) of PHE 2.5 mg/Kg treated rats. (*** P<0.001) Taken together, these data showed an increase in VEGF expression in low urothelium from PHE-treated rats, when compared to high urothelium from PHE-treated and the one of control rats. 0 200 400 600 Control PHE (high uro) PHE (low uro) Signal intensity *** A B C 21 Expression of PlGF in the urothelium PlGF belongs to the VEGF family, and expression might be altered due to the chronic adrenergic stimulation. In this experience, we aimed at studying the expression of PlGF, in the urothelium from control and PHE-treated rats. PlGF levels were analysed by immunohistochemistry. Intensity signal of PlGF immunoreaction (PlGF-IR) in the urinary bladder of control animals was 45 +/- 22 (Figure 4A and 4C). In PHE-treated animals, the signal intensity of PlGF-IR was 64 +/- 11 in normal urothelium (Figure 4B, black arrows, and 4C) and 370 +/- 248 in regions with low urothelium (Figure 4B, red arrow, and 4C). Figure 4: Photomicrographs of PlGF expression in the urothelium of control rats (A) and in high (B, dark arrows) and low urothelium (B, red arrow) from PHE 2.5 mg/Kg -treated rats. C: Mean signal intensity of PlGF expression in the urothelium of a control and in the normal urothelium areas (high uro) and low urothelium areas (low uro) of PHE 2.5 mg/Kg treated rats. (*** P<0.001) Together, these data show an augment in PlGF expression in low urothelium from PHE-treated rats, when compared to high urothelium of PHE-treated rats and the urothelium from control rats. 0 200 400 600 800 Control PHE (high uro) PHE (low uro) Signal Intensity *** A B C 22 Expression of VEGFR1 in the urothelium In this experience, we intended to analyse the expression of the receptor for VEGF and PlGF, VEGFR1, in the urothelium from control and PHE-treated rats. VEGFR1 levels were determined by immunohistochemistry. Intensity signal of VEGFR1 immunoreaction (VEGFR1-IR) in the urinary bladder of control animals was 102 +/- 4 (Figure 5A and 5D). In PHE-treated animals, the signal intensity of VEGFR1-IR was 107 +/- 5 in normal urothelium (Figure 5B and 5D) and 128 +/- 9 in regions with low urothelium (Figure 5B and 5D). Figure 5: Photomicrographs of VEGFR1 expression in the urothelium of control rats (A) and in high (B) and low urothelium (C) from PHE 2.5 mg/Kg -treated rats. D: Mean signal intensity of VEGFR1 expression in the urothelium of a control and in the normal urothelium areas (high uro) and low urothelium areas (low uro) of PHE 2.5 mg/Kg treated rats. (*** P<0.001) VEGFR1 expression was higher in low urothelium from PHE-treated rats, when compared to high urothelium of PHE-treated rats and the urothelium from control rats. 0 20 40 60 80 100 120 140 160 Control PHE (Uro High) PHE (Uro Low) Signal Intensity *** A B C 29 VEGF quantification in urine Urinary VEGF levels obtained for both of the pools of urine (PHE-treated rats and control rats) were inconclusive. There were only two pools of urine (urine from control rats versus urine from treated rats). The volume of urine collected from each rat was reduced, so two pools were made from each group. One solution to make the results statistically significant is to collect more samples of urine from a major number of rats, control and PHE-treated rats, and organize them in more pools. 30 Conclusions 31 Conclusions The present work showed that chronic treatment with phenylephrine in female rats caused the overexpression of VEGF, PlGF and VEGFR1 in the low bladder urothelium. One can hypothesize that VEGF and PlGF acting on VEGFR1 may induce urothelial degradation, by activating apoptotic enzymes. Chronic adrenergic stimulation did not induce any change in VEGF, PlGF and VEGFR-1 expression in the detrusor smooth muscle, suggesting that VEGF related molecules are not involved in direct control of detrusor activity during chronic adrenergic stimulation. The results obtained for VEGF concentration in urine from PHE-treated and control rats were inconclusive. A major number of samples should be assessed in order to understand if VEGF levels changes upon adrenergic stimulation. 32 Future Prospects 33 Future Prospects Further approaches should be done in order to understand the true role of VEGF in BPS/IC. VEGF might be involved in inflammatory, angiogenic, apoptotic and chemotactic processes in BPS/IC. Previous studies in PHE-chronically treated rats showed the occurrence of apoptotic in the urothelium and bladder inflammation. The present study raises the hypothesis that VEGF related molecules may be involved in these mechanisms. Therefore, co-localization of VEGF and its related molecules and apoptotic enzymes should be addressed. Furthermore, experiment conduction to the confirmation of VEGF chemotaxis role should be addressed. Although chronic adrenergic stimulation does not induce any macroscopic changes in the urinary bladder, such as petechial appearance, angiogenesis can be addressed by studying pericyte migration and new vessel forming. VEGF signalling should be studied at a molecular level in this model in order to fully understand the apoptotic and angiogenic events, for instance involvement of Akt/PKB or PKC, which may explain the apoptosis, and relation between VEGFR1 and VEGFR2. 34 Acknowledgements 35 Acknowledgements The ship has reached the end of the journey. It went through numerous storms and also some bright days. I was just a humble sailor commanded by a captain, the one that was capable of doing the route planning and suggested solutions to the problems I tried to overcome. Professor Francisco Cruz was the mentor of the idea, and Professor Ana Charrua, my captain, and Professor Célia Cruz were there giving me the right instructions to take the boat to a safe harbour. I owe them special thanks because of the opportunity they gave me. It was an enormous privilege to me. The other members of the crew, who were sailors with more experience, gave me help and guided me along the way, teaching me the best methods to carry on with my work. I would like to thank MSci Bárbara Frias, Professor António Avelino, MSci Sérgio Barros, Anabela Silvestre, Ana Pessoa and MSci Inês Tomada for being so kind and helpful. During the journey, I met kind and funny people like Miguel, César, Isabel, Mariana, Diana, Telmo, José Pedro, Palmira, Ana Coelho, Margarida Oliveira from Department of Experimental Biology and Cátia Vieira from Department of Pharmacology in ICBAS and had nice meals and coffees with them. I thank Mrs. Glória Martins, Mrs Elisa Nova, Mr. Fernando Martins, Mrs. Fátima Moreira, Mrs. Raquel Madanços, Mrs. Ana Tavares and Marta Louçano for their kindness. And also, I owe a thank to all of the other collaborators from the Department of Experimental Biology, including Professor Castro Lopes, Professor Fani Neto, Professor Alexandra Gouveia , MSci Liliana Matos, Adriana Rodrigues, Isabel Martins, Daniel Martins, Professor Daniel Pozza, Professor Carlos Reguenga, Professor Filipe Monteiro, MSci Sara Adães, and Professor Delminda Magalhães. And Professor Deolinda Lima, for being such a wonderful leader of this Department. I hope I am not forgetting anyone. When the ship was about to sink and I was losing hope, there were some friends to back me up. Raquel Oliveira, my lab mate, was there listening to my day-to-day problems and so did my master friends, Marisa Coelho and Cátia Costa. Long-date friends Gabriela Albuquerque, Ana Margarida Martins, Lucília Silva and Luís Silva were always worried about my success in this Project, and never gave up on believing that I was capable of finishing this work. I thank them for everything. I also need to thank my other friends from Guimarães, Sociedade de Debates da Universidade do Porto and from the different faculties from University of Porto for these academic years that went by. Thank you, Nuno Lamas, for your time and help. Your wise advices were certainly taken into account. 36 My parents and my sister Cláudia deserve the biggest thanks for believing in me during all these 24 years (Cláudia, just during 22 till now, because she is my lil’ sis), and made all of my academic studies possible. I hope to not disappoint them in any time, because from now on, I think it is my time for providing them the best and making them proud. And I am deeply grateful to Artur. You know what you mean to me. 37 Bibliography 38 Bibliography Birder, L. A., Hanna-Mitchell, A. T., Mayer, E., & Buffington, C. A. . Cystitis, comorbid disorders and associated epithelial dysfunction. Neurourology and urodynamics, 30(5), 2011, 668–672 Charrua, A., Pinto, R., Taylor, A. 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