Internal standard-based analysis of microarray data2--analysis of functional associations between HVE-genes.

In this work we apply the Internal Standard-based analytical approach that we described in an earlier communication and here we demonstrate experimental results on functional associations among the hypervariably-expressed genes (HVE-genes). Our working assumption was that those genetic components, which initiate the disease, involve HVE-genes for which the level of expression is undistinguishable among healthy individuals and individuals with pathology. We show that analysis of the functional associations of the HVE-genes is indeed suitable to revealing disease-specific differences. We show also that another possible exploit of HVE-genes for characterization of pathological alterations is by using multivariate classification methods. This in turn offers important clues on naturally occurring dynamic processes in the organism and is further used for dynamic discrimination of groups of compared samples. We conclude that our approach can uncover principally new collective differences that cannot be discerned by individual gene analysis.

VEGF induces sensory and motor peripheral plasticity, alters bladder function, and promotes visceral sensitivity.

BACKGROUND: This work tests the hypothesis that bladder instillation with vascular endothelial growth factor (VEGF) modulates sensory and motor nerve plasticity, and, consequently, bladder function and visceral sensitivity.In addition to C57BL/6J, ChAT-cre mice were used for visualization of bladder cholinergic nerves. The direct effect of VEGF on the density of sensory nerves expressing the transient receptor potential vanilloid subfamily 1 (TRPV1) and cholinergic nerves (ChAT) was studied one week after one or two intravesical instillations of the growth factor.To study the effects of VEGF on bladder function, mice were intravesically instilled with VEGF and urodynamic evaluation was assessed. VEGF-induced alteration in bladder dorsal root ganglion (DRG) neurons was performed on retrogradly labeled urinary bladder afferents by patch-clamp recording of voltage gated Na+ currents. Determination of VEGF-induced changes in sensitivity to abdominal mechanostimulation was performed by application of von Frey filaments. RESULTS: In addition to an overwhelming increase in TRPV1 immunoreactivity, VEGF instillation resulted in an increase in ChAT-directed expression of a fluorescent protein in several layers of the urinary bladder. Intravesical VEGF caused a profound change in the function of the urinary bladder: acute VEGF (1 week post VEGF treatment) reduced micturition pressure and longer treatment (2 weeks post-VEGF instillation) caused a substantial reduction in inter-micturition interval. In addition, intravesical VEGF resulted in an up-regulation of voltage gated Na(+) channels (VGSC) in bladder DRG neurons and enhanced abdominal sensitivity to mechanical stimulation. CONCLUSIONS: For the first time, evidence is presented indicating that VEGF instillation into the mouse bladder promotes a significant increase in peripheral nerve density together with alterations in bladder function and visceral sensitivity. The VEGF pathway is being proposed as a key modulator of neural plasticity in the pelvis and enhanced VEGF content may be associated with visceral hyperalgesia, abdominal discomfort, and/or pelvic pain.

VEGF signaling mediates bladder neuroplasticity and inflammation in response to BCG.

BACKGROUND: This work tests the hypothesis that increased levels of vascular endothelial growth factor (VEGF) observed during bladder inflammation modulates nerve plasticity. METHODS: Chronic inflammation was induced by intravesical instillations of Bacillus Calmette-Guérin (BCG) into the urinary bladder and the density of nerves expressing the transient receptor potential vanilloid subfamily 1 (TRPV1) or pan-neuronal marker PGP9.5 was used to quantify alterations in peripheral nerve plasticity. Some mice were treated with B20, a VEGF neutralizing antibody to reduce the participation of VEGF. Additional mice were treated systemically with antibodies engineered to specifically block the binding of VEGF to NRP1 (anti-NRP1B) and NRP2 (NRP2B), or the binding of semaphorins to NRP1 (anti-NRP1 A) to diminish activity of axon guidance molecules such as neuropilins (NRPs) and semaphorins (SEMAs). To confirm that VEGF is capable of inducing inflammation and neuronal plasticity, another group of mice was instilled with recombinant VEGF165 or VEGF121 into the urinary bladder. RESULTS: The major finding of this work was that chronic BCG instillation resulted in inflammation and an overwhelming increase in both PGP9.5 and TRPV1 immunoreactivity, primarily in the sub-urothelium of the urinary bladder. Treatment of mice with anti-VEGF neutralizing antibody (B20) abolished the effect of BCG on inflammation and nerve density.NRP1A and NRP1B antibodies, known to reduce BCG-induced inflammation, failed to block BCG-induced increase in nerve fibers. However, the NRP2B antibody dramatically potentiated the effects of BCG in increasing PGP9.5-, TRPV1-, substance P (SP)-, and calcitonin gene-related peptide (CGRP)-immunoreactivity (IR). Finally, instillation of VEGF121 or VEGF165 into the mouse bladder recapitulated the effects of BCG and resulted in a significant inflammation and increase in nerve density. CONCLUSIONS: For the first time, evidence is being presented supporting that chronic BCG instillation into the mouse bladder promotes a significant increase in peripheral nerve density that was mimicked by VEGF instillation. Effects of BCG were abolished by pre-treatment with neutralizing VEGF antibody. The present results implicate the VEGF pathway as a key modulator of inflammation and nerve plasticity, introduces a new animal model for investigation of VEGF-induced nerve plasticity, and suggests putative mechanisms underlying this phenomenon.

Neuropilin-VEGF signaling pathway acts as a key modulator of vascular, lymphatic, and inflammatory cell responses of the bladder to intravesical BCG treatment.

Recent findings indicate that VEGF receptors and coreceptors (neuropilins; NRP) are expressed on nonendothelial cells in human bladder urothelium, in one human bladder cancer cell line (J82), and in the mouse bladder urothelium. In addition, VEGFR1, VEGFR2, NRP1, and NRP2 expressions were upregulated in animal models of chronic bladder inflammation induced by four weekly instillations of protease-activated receptors (PAR)-activating peptides or bacillus Calmette-Guérin (BCG) into the mouse bladder. Here, we used four weekly instillations of BCG as a model for chronic bladder inflammation to further investigate whether VEGF receptors and NRPs play a role in the migration of inflammatory cells and inflammation-induced lymphangiogenesis and angiogenesis. For this purpose, we used neutralizing antibodies that were engineered to specifically block the binding of VEGF to NRP (anti-NRP1(B)) and the binding of semaphorins to NRP (anti-NRP1(A)). C57BL/6 mice received intraperitoneal injections of PBS, anti-NRP1(A)- or anti-NRP1(B)-neutralizing antibodies and then were challenged chronically with intravesical PBS or BCG. At the end of chronic challenge period, a fluorescent internalizable tracer, scVEGF/Cy5.5, was administered to all mice and near-infrared fluorescence images were obtained in vivo and in real time. BCG increased the overall accumulation of scVEGF/Cy5.5 in the urinary bladder urothelium and inflammatory cells. In addition, BCG increased the density of blood and lymphatic vessels concomitantly with an upregulation of NRP2 expression in lymphatic vessels. Treatment of the mice with NRP1-neutralizing antibodies dramatically reduced scVEGF/Cy5.5 uptake, polymorphonuclear (myeloperoxidase-positive cells) and dendritic cell (CD11c-positive cells) infiltration, and decreased the overall density of BCG-induced blood and lymphatic vessels. These results implicate NRPs as critical in vivo regulators of the vascular and inflammatory responses to the intravesical administration of BCG.

Frankincense oil derived from Boswellia carteri induces tumor cell specific cytotoxicity.

BACKGROUND: Originating from Africa, India, and the Middle East, frankincense oil has been important both socially and economically as an ingredient in incense and perfumes for thousands of years. Frankincense oil is prepared from aromatic hardened gum resins obtained by tapping Boswellia trees. One of the main components of frankincense oil is boswellic acid, a component known to have anti-neoplastic properties. The goal of this study was to evaluate frankincense oil for its anti-tumor activity and signaling pathways in bladder cancer cells. METHODS: Frankincense oil-induced cell viability was investigated in human bladder cancer J82 cells and immortalized normal bladder urothelial UROtsa cells. Temporal regulation of frankincense oil-activated gene expression in bladder cancer cells was identified by microarray and bioinformatics analysis. RESULTS: Within a range of concentration, frankincense oil suppressed cell viability in bladder transitional carcinoma J82 cells but not in UROtsa cells. Comprehensive gene expression analysis confirmed that frankincense oil activates genes that are responsible for cell cycle arrest, cell growth suppression, and apoptosis in J82 cells. However, frankincense oil-induced cell death in J82 cells did not result in DNA fragmentation, a hallmark of apoptosis. CONCLUSION: Frankincense oil appears to distinguish cancerous from normal bladder cells and suppress cancer cell viability. Microarray and bioinformatics analysis proposed multiple pathways that can be activated by frankincense oil to induce bladder cancer cell death. Frankincense oil might represent an alternative intravesical agent for bladder cancer treatment.

From microarray to biology: an integrated experimental, statistical and in silico analysis of how the extracellular matrix modulates the phenotype of cancer cells.

A statistically robust and biologically-based approach for analysis of microarray data is described that integrates independent biological knowledge and data with a global F-test for finding genes of interest that minimizes the need for replicates when used for hypothesis generation. First, each microarray is normalized to its noise level around zero. The microarray dataset is then globally adjusted by robust linear regression. Second, genes of interest that capture significant responses to experimental conditions are selected by finding those that express significantly higher variance than those expressing only technical variability. Clustering expression data and identifying expression-independent properties of genes of interest including upstream transcriptional regulatory elements (TREs), ontologies and networks or pathways organizes the data into a biologically meaningful system. We demonstrate that when the number of genes of interest is inconveniently large, identifying a subset of "beacon genes" representing the largest changes will identify pathways or networks altered by biological manipulation. The entire dataset is then used to complete the picture outlined by the "beacon genes." This allow construction of a structured model of a system that can generate biologically testable hypotheses. We illustrate this approach by comparing cells cultured on plastic or an extracellular matrix which organizes a dataset of over 2,000 genes of interest from a genome wide scan of transcription. The resulting model was confirmed by comparing the predicted pattern of TREs with experimental determination of active transcription factors.

Molecular networks discriminating mouse bladder responses to intravesical bacillus Calmette-Guerin (BCG), LPS, and TNF-alpha.

BACKGROUND: Despite being a mainstay for treating superficial bladder carcinoma and a promising agent for interstitial cystitis, the precise mechanism of Bacillus Calmette-Guerin (BCG) remains poorly understood. It is particularly unclear whether BCG is capable of altering gene expression in the bladder target organ beyond its well-recognized pro-inflammatory effects and how this relates to its therapeutic efficacy. The objective of this study was to determine differentially expressed genes in the mouse bladder following chronic intravesical BCG therapy and to compare the results to non-specific pro inflammatory stimuli (LPS and TNF-alpha). For this purpose, C57BL/6 female mice received four weekly instillations of BCG, LPS, or TNF-alpha. Seven days after the last instillation, the urothelium along with the submucosa was removed from detrusor muscle and the RNA was extracted from both layers for cDNA array experiments. Microarray results were normalized by a robust regression analysis and only genes with an expression above a conditional threshold of 0.001 (3SD above background) were selected for analysis. Next, genes presenting a 3-fold ratio in regard to the control group were entered in Ingenuity Pathway Analysis (IPA) for a comparative analysis in order to determine genes specifically regulated by BCG, TNF-alpha, and LPS. In addition, the transcriptome was precipitated with an antibody against RNA polymerase II and real-time polymerase chain reaction assay (Q-PCR) was used to confirm some of the BCG-specific transcripts. RESULTS: Molecular networks of treatment-specific genes generated several hypotheses regarding the mode of action of BCG. BCG-specific genes involved small GTPases and BCG-specific networks overlapped with the following canonical signaling pathways: axonal guidance, B cell receptor, aryl hydrocarbon receptor, IL-6, PPAR, Wnt/beta-catenin, and cAMP. In addition, a specific detrusor network expressed a high degree of overlap with the development of the lymphatic system. Interestingly, TNF-alpha-specific networks overlapped with the following canonical signaling pathways: PPAR, death receptor, and apoptosis. Finally, LPS-specific networks overlapped with the LPS/IL-1 mediated inhibition of RXR. Because NF-kappaB occupied a central position in several networks, we further determined whether this transcription factor was part of the responses to BCG. Electrophoretic mobility shift assays confirmed the participation of NF-kappaB in the mouse bladder responses to BCG. In addition, BCG treatment of a human urothelial cancer cell line (J82) also increased the binding activity of NF-kappaB, as determined by precipitation of the chromatin by a NF-kappaB-p65 antibody and Q-PCR of genes bearing a NF-kappaB consensus sequence. Next, we tested the hypothesis of whether small GTPases such as LRG-47 are involved in the uptake of BCG by the bladder urothelium. CONCLUSION: As expected, BCG treatment induces the transcription of genes belonging to common pro-inflammatory networks. However, BCG also induces unique genes belonging to molecular networks involved in axonal guidance and lymphatic system development within the bladder target organ. In addition, NF-kappaB seems to play a predominant role in the bladder responses to BCG therapy. Finally, in intact urothelium, BCG-GFP internalizes in LRG-47-positive vesicles. These results provide a molecular framework for the further study of the involvement of immune and nervous systems in the bladder responses to BCG therapy.

Systems biology approach for mapping the response of human urothelial cells to infection by Enterococcus faecalis.

BACKGROUND: To better understand the response of urinary epithelial (urothelial) cells to Enterococcus faecalis, a uropathogen that exhibits resistance to multiple antibiotics, a genome-wide scan of gene expression was obtained as a time series from urothelial cells growing as a layered 3-dimensional culture similar to normal urothelium. We herein describe a novel means of analysis that is based on deconvolution of gene variability into technical and biological components. RESULTS: Analysis of the expression of 21,521 genes from 30 minutes to 10 hours post infection, showed 9553 genes were expressed 3 standard deviations (SD) above the system zero-point noise in at least 1 time point. The asymmetric distribution of relative variances of the expressed genes was deconvoluted into technical variation (with a 6.5% relative SD) and biological variation components (>3 SD above the mode technical variability). These 1409 hypervariable (HV) genes encapsulated the effect of infection on gene expression. Pathway analysis of the HV genes revealed an orchestrated response to infection in which early events included initiation of immune response, cytoskeletal rearrangement and cell signaling followed at the end by apoptosis and shutting down cell metabolism. The number of poorly annotated genes in the earliest time points suggests heretofore unknown processes likely also are involved. CONCLUSION: Enterococcus infection produced an orchestrated response by the host cells involving several pathways and transcription factors that potentially drive these pathways. The early time points potentially identify novel targets for enhancing the host response. These approaches combine rigorous statistical principles with a biological context and are readily applied by biologists.

Discriminators of mouse bladder response to intravesical Bacillus Calmette-Guerin (BCG).

BACKGROUND: Intravesical Bacillus Calmette-Guerin (BCG) is an effective treatment for bladder superficial carcinoma and it is being tested in interstitial cystitis patients, but its precise mechanism of action remains poorly understood. It is not clear whether BCG induces the release of a unique set of cytokines apart from its pro-inflammatory effects. Therefore, we quantified bladder inflammatory responses and alterations in urinary cytokine protein induced by intravesical BCG and compared the results to non-specific pro-inflammatory stimuli (LPS and TNF-alpha). We went further to determine whether BCG treatment alters cytokine gene expression in the urinary bladder. METHODS: C57BL/6 female mice received four weekly instillations of BCG, LPS, or TNF-alpha. Morphometric analyses were conducted in bladders isolated from all groups and urine was collected for multiplex analysis of 18 cytokines. In addition, chromatin immune precipitation combined with real-time polymerase chain reaction assay (CHIP/Q-PCR) was used to test whether intravesical BCG would alter bladder cytokine gene expression. RESULTS: Acute BCG instillation induced edema which was progressively replaced by an inflammatory infiltrate, composed primarily of neutrophils, in response to weekly administrations. Our morphological analysis suggests that these polymorphonuclear neutrophils are of prime importance for the bladder responses to BCG. Overall, the inflammation induced by BCG was higher than LPS or TNF-alpha treatment but the major difference observed was the unique granuloma formation in response to BCG. Among the cytokines measured, this study highlighted the importance of IL-1beta, IL-2, IL-3, IL-4, IL-6, IL-10, IL-17, GM-CSF, KC, and Rantes as discriminators between generalized inflammation and BCG-specific inflammatory responses. CHIP/Q-PCR indicates that acute BCG instillation induced an up-regulation of IL-17A, IL-17B, and IL-17RA, whereas chronic BCG induced IL-17B, IL-17RA, and IL-17RB. CONCLUSION: To the best of our knowledge, the present work is the first to report that BCG induces an increase in the IL-17 family genes. In addition, BCG induces a unique type of persisting bladder inflammation different from TNF-alpha, LPS, and, most likely, other classical pro-inflammatory stimuli.

Transcription factor network downstream of protease activated receptors (PARs) modulating mouse bladder inflammation.

BACKGROUND: All four PARs are present in the urinary bladder, and their expression is altered during inflammation. In order to search for therapeutic targets other than the receptors themselves, we set forth to determine TFs downstream of PAR activation in the C57BL/6 urinary bladders. METHODS: For this purpose, we used a protein/DNA combo array containing 345 different TF consensus sequences. Next, the TF selected was validated by EMSA and IHC. As mast cells seem to play a fundamental role in bladder inflammation, we determined whether c-kit receptor deficient (Kit w/Kit w-v) mice have an abrogated response to PAR stimulation. Finally, TFEB antibody was used for CHIP/Q-PCR assay and revealed up-regulation of genes known to be downstream of TFEB. RESULTS: TFEB, a member of the MiTF family of basic helix-loop-helix leucine zipper, was the only TF commonly up-regulated by all PAR-APs. IHC results confirm a correlation between inflammation and TFEB expression in C57BL/6 mice. In contrast, Kit w/Kit w-v mice did not exhibit inflammation in response to PAR activation. EMSA results confirmed the increased TFEB binding activity in C57BL/6 but not in Kit w/Kit w-v mice. CONCLUSION: This is the first report describing the increased expression of TFEB in bladder inflammation in response to PAR activation. As TFEB belongs to a family of TFs essential for mast cell survival, our findings suggest that this molecule may influence the participation of mast cells in PAR-mediated inflammation and that targeting TFEB/MiTF activity may be a novel approach for the treatment of bladder inflammatory disorders.

Lymphatic vessel density and function in experimental bladder cancer.

BACKGROUND: The lymphatics form a second circulatory system that drains the extracellular fluid and proteins from the tumor microenvironment, and provides an exclusive environment in which immune cells interact and respond to foreign antigen. Both cancer and inflammation are known to induce lymphangiogenesis. However, little is known about bladder lymphatic vessels and their involvement in cancer formation and progression. METHODS: A double transgenic mouse model was generated by crossing a bladder cancer-induced transgenic, in which SV40 large T antigen was under the control of uroplakin II promoter, with another transgenic mouse harboring a lacZ reporter gene under the control of an NF-kappaB-responsive promoter (kappaB-lacZ) exhibiting constitutive activity of beta-galactosidase in lymphatic endothelial cells. In this new mouse model (SV40-lacZ), we examined the lymphatic vessel density (LVD) and function (LVF) during bladder cancer progression. LVD was performed in bladder whole mounts and cross-sections by fluorescent immunohistochemistry (IHC) using LYVE-1 antibody. LVF was assessed by real-time in vivo imaging techniques using a contrast agent (biotin-BSA-Gd-DTPA-Cy5.5; Gd-Cy5.5) suitable for both magnetic resonance imaging (MRI) and near infrared fluorescence (NIRF). In addition, IHC of Cy5.5 was used for time-course analysis of co-localization of Gd-Cy5.5 with LYVE-1-positive lymphatics and CD31-positive blood vessels. RESULTS: SV40-lacZ mice develop bladder cancer and permitted visualization of lymphatics. A significant increase in LVD was found concomitantly with bladder cancer progression. Double labeling of the bladder cross-sections with LYVE-1 and Ki-67 antibodies indicated cancer-induced lymphangiogenesis. MRI detected mouse bladder cancer, as early as 4 months, and permitted to follow tumor sizes during cancer progression. Using Gd-Cy5.5 as a contrast agent for MRI-guided lymphangiography, we determined a possible reduction of lymphatic flow within the tumoral area. In addition, NIRF studies of Gd-Cy5.5 confirmed its temporal distribution between CD31-positive blood vessels and LYVE-1 positive lymphatic vessels. CONCLUSION: SV40-lacZ mice permit the visualization of lymphatics during bladder cancer progression. Gd-Cy5.5, as a double contrast agent for NIRF and MRI, permits to quantify delivery, transport rates, and volumes of macromolecular fluid flow through the interstitial-lymphatic continuum. Our results open the path for the study of lymphatic activity in vivo and in real time, and support the role of lymphangiogenesis during bladder cancer progression.

Repeated BCG treatment of mouse bladder selectively stimulates small GTPases and HLA antigens and inhibits single-spanning uroplakins.

BACKGROUND: Despite being a mainstay for treating superficial bladder carcinoma and a promising agent for interstitial cystitis, the precise mechanism of Bacillus Calmette-Guerin (BCG) remains poorly understood. It is particularly unclear whether BCG is capable of altering gene expression beyond its well-recognized pro-inflammatory effects and how this relates to its therapeutic efficacy. The objective of this study was to determine differentially expressed genes in the mouse bladder following repeated intravesical BCG therapy. METHODS: Mice were transurethrally instilled with BCG or pyrogen-free on days 1, 7, 14, and 21. Seven days after the last instillation, urothelia along with the submucosa was removed and amplified ds-DNA was prepared from control- and BCG-treated bladder mucosa and used to generate suppression subtractive hybridization (SSH). Plasmids from control- and BCG-specific differentially expressed clones and confirmed by Virtual Northern were then purified and the inserts were sequenced and annotated. Finally, chromatin immune precipitation combined with real-time polymerase chain reaction assay (ChIP/Q-PCR) was used to validate SSH-selected transcripts. RESULTS: Repeated intravesical BCG treatment induced an up regulation of genes associated with antigen presentation (B2M, HLA-A, HLA-DQA1, HLA-DQB2, HLA-E, HLA-G, IGHG, and IGH) and representatives of two IFNgamma-induced small GTPase families: the GBPs (GBP1, GBP2, and GBP5) and the p47GTPases (IIGTP1, IIGTP2, and TGTP). Genes expressed in saline-treated bladders but down-regulated by BCG included: the single-spanning uroplakins (UPK3a and UPK2), SPRR2G, GSTM5, and RSP 19. CONCLUSION: Here we introduced a hypothesis-generator approach to determine key genes involved in the urothelium/sumbmucosa responses to BCG therapy. Urinary bladder responds to repeated BCG treatment by up-regulating not only antigen presentation-related genes, but also GBP and p47 small GTPases, both potentially serving to mount a resistance to the replication of the Mycobacterium. It will be of tremendous future interest to determine whether these immune response cascades play a role in the anti-cancer effects exerted by BCG.

Regulatory network of inflammation downstream of proteinase-activated receptors.

BACKGROUND: Protease-activated receptors (PAR) are present in the urinary bladder, and their expression is altered in response to inflammation. PARs are a unique class of G protein-coupled that carry their own ligands, which remain cryptic until unmasked by proteolytic cleavage. Although the canonical signal transduction pathway downstream of PAR activation and coupling with various G proteins is known and leads to the rapid transcription of genes involved in inflammation, the effect of PAR activation on the downstream transcriptome is unknown. We have shown that intravesical administration of PAR-activating peptides leads to an inflammatory reaction characterized by edema and granulocyte infiltration. Moreover, the inflammatory response to intravesical instillation of known pro-inflammatory stimuli such as E. coli lipopolysaccharide (LPS), substance P (SP), and antigen was strongly attenuated by PAR1- and to a lesser extent by PAR2-deficiency. RESULTS: Here, cDNA array experiments determined inflammatory genes whose expression is dependent on PAR1 activation. For this purpose, we compared the alteration in gene expression in wild type and PAR1-/- mice induced by classical pro-inflammatory stimuli (LPS, SP, and antigen). 75 transcripts were considered to be dependent on PAR-1 activation and further annotated in silico by Ingenuity Pathways Analysis (IPA) and gene ontology (GO). Selected transcripts were target validated by quantitative PCR (Q-PCR). Among PAR1-dependent transcripts, the following have been implicated in the inflammatory process: b2m, ccl7, cd200, cd63, cdbpd, cfl1, dusp1, fkbp1a, fth1, hspb1, marcksl1, mmp2, myo5a, nfkbia, pax1, plaur, ppia, ptpn1, ptprcap, s100a10, sim2, and tnfaip2. However, a balanced response to signals of injury requires a transient cellular activation of a panel of genes together with inhibitory systems that temper the overwhelming inflammation. In this context, the activation of genes such as dusp1 and nfkbia seems to counter-balance the inflammatory response to PAR activation by limiting prolonged activation of p38 MAPK and increased cytokine production. In contrast, transcripts such as arf6 and dcnt1 that are involved in the mechanism of PAR re-sensitization would tend to perpetuate the inflammatory reaction in response to common pro-inflammatory stimuli. CONCLUSION: The combination of cDNA array results and genomic networks reveals an overriding participation of PAR1 in bladder inflammation, provides a working model for the involvement of downstream signaling, and evokes testable hypotheses regarding the transcriptome downstream of PAR1 activation. It remains to be determined whether or not mechanisms targeting PAR1 gene silencing or PAR1 blockade will ameliorate the clinical manifestation of cystitis.

Mandatory role of proteinase-activated receptor 1 in experimental bladder inflammation.

BACKGROUND: In general, inflammation plays a role in most bladder pathologies and represents a defense reaction to injury that often times is two edged. In particular, bladder neurogenic inflammation involves the participation of mast cells and sensory nerves. Increased mast cell numbers and tryptase release represent one of the prevalent etiologic theories for interstitial cystitis and other urinary bladder inflammatory conditions. The activity of mast cell-derived tryptase as well as thrombin is significantly increased during inflammation. Those enzymes activate specific G-protein coupled proteinase-activated receptors (PAR)s. Four PARs have been cloned so far, and not only are all four receptors highly expressed in different cell types of the mouse urinary bladder, but their expression is altered during experimental bladder inflammation. We hypothesize that PARs may link mast cell-derived proteases to bladder inflammation and, therefore, play a fundamental role in the pathogenesis of cystitis. RESULTS: Here, we demonstrate that in addition to the mouse urinary bladder, all four PA receptors are also expressed in the J82 human urothelial cell line. Intravesical administration of PAR-activating peptides in mice leads to an inflammatory reaction characterized by edema and granulocyte infiltration. Moreover, the inflammatory response to intravesical instillation of known pro-inflammatory stimuli such as E. coli lipopolysaccharide (LPS), substance P, and antigen was strongly attenuated by PAR1-, and to a lesser extent, by PAR2-deficiency. CONCLUSION: Our results reveal an overriding participation of PAR1 in bladder inflammation, provide a working model for the involvement of downstream signaling, and evoke testable hypotheses regarding the role of PARs in bladder inflammation. It remains to be determined whether or not mechanisms targeting PAR1 gene silencing or PAR1 blockade will ameliorate the clinical manifestations of cystitis.

Bladder inflammatory transcriptome in response to tachykinins: neurokinin 1 receptor-dependent genes and transcription regulatory elements.

BACKGROUND: Tachykinins (TK), such as substance P, and their neurokinin receptors which are ubiquitously expressed in the human urinary tract, represent an endogenous system regulating bladder inflammatory, immune responses, and visceral hypersensitivity. Increasing evidence correlates alterations in the TK system with urinary tract diseases such as neurogenic bladders, outflow obstruction, idiopathic detrusor instability, and interstitial cystitis. However, despite promising effects in animal models, there seems to be no published clinical study showing that NK-receptor antagonists are an effective treatment of pain in general or urinary tract disorders, such as detrusor overactivity. In order to search for therapeutic targets that could block the tachykinin system, we set forth to determine the regulatory network downstream of NK1 receptor activation. First, NK1R-dependent transcripts were determined and used to query known databases for their respective transcription regulatory elements (TREs). METHODS: An expression analysis was performed using urinary bladders isolated from sensitized wild type (WT) and NK1R-/- mice that were stimulated with saline, LPS, or antigen to provoke inflammation. Based on cDNA array results, NK1R-dependent genes were selected. PAINT software was used to query TRANSFAC database and to retrieve upstream TREs that were confirmed by electrophoretic mobility shift assays. RESULTS: The regulatory network of TREs driving NK1R-dependent genes presented cRel in a central position driving 22% of all genes, followed by AP-1, NF-kappaB, v-Myb, CRE-BP1/c-Jun, USF, Pax-6, Efr-1, Egr-3, and AREB6. A comparison between NK1R-dependent and NK1R-independent genes revealed Nkx-2.5 as a unique discriminator. In the presence of NK1R, Nkx2-5 _01 was significantly correlated with 36 transcripts which included several candidates for mediating bladder development (FGF) and inflammation (PAR-3, IL-1R, IL-6, alpha-NGF, TSP2). In the absence of NK1R, the matrix Nkx2-5_02 had a predominant participation driving 8 transcripts, which includes those involved in cancer (EYA1, Trail, HSF1, and ELK-1), smooth-to-skeletal muscle trans-differentiation, and Z01, a tight-junction protein, expression. Electrophoretic mobility shift assays confirmed that, in the mouse urinary bladder, activation of NK1R by substance P (SP) induces both NKx-2.5 and NF-kappaB translocations. CONCLUSION: This is the first report describing a role for Nkx2.5 in the urinary tract. As Nkx2.5 is the unique discriminator of NK1R-modulated inflammation, it can be imagined that in the near future, new based therapies selective for controlling Nkx2.5 activity in the urinary tract may be used in the treatment in a number of bladder disorders.

Analysis of the interaction of extracellular matrix and phenotype of bladder cancer cells.

BACKGROUND: The extracellular matrix has a major effect upon the malignant properties of bladder cancer cells both in vitro in 3-dimensional culture and in vivo. Comparing gene expression of several bladder cancer cells lines grown under permissive and suppressive conditions in 3-dimensional growth on cancer-derived and normal-derived basement membrane gels respectively and on plastic in conventional tissue culture provides a model system for investigating the interaction of malignancy and extracellular matrix. Understanding how the extracellular matrix affects the phenotype of bladder cancer cells may provide important clues to identify new markers or targets for therapy. METHODS: Five bladder cancer cell lines and one immortalized, but non-tumorigenic, urothelial line were grown on Matrigel, a cancer-derived ECM, on SISgel, a normal-derived ECM, and on plastic, where the only ECM is derived from the cells themselves. The transcriptomes were analyzed on an array of 1186 well-annotated cancer derived cDNAs containing most of the major pathways for malignancy. Hypervariable genes expressing more variability across cell lines than a set expressing technical variability were analyzed further. Expression values were clustered, and to identify genes most likely to represent biological factors, statistically over-represented ontologies and transcriptional regulatory elements were identified. RESULTS: Approximately 400 of the 1186 total genes were expressed 2 SD above background. Approximately 100 genes were hypervariable in cells grown on each ECM, but the pattern was different in each case. A core of 20 were identified as hypervariable under all 3 growth conditions, and 33 were hypervariable on both SISgel and Matrigel, but not on plastic. Clustering of the hypervariable genes showed very different patterns for the same 6 cell types on the different ECM. Even when loss of cell cycle regulation was identified, different genes were involved, depending on the ECM. Under the most permissive conditions of growth where the malignant phenotype was fully expressed, activation of AKT was noted. TGFbeta1 signaling played a major role in the response of bladder cancer cells to ECM. Identification of TREs on genes that clustered together suggested some clustering was driven by specific transcription factors. CONCLUSION: The extracellular matrix on which cancer cells are grown has a major effect on gene expression. A core of 20 malignancy-related genes were not affected by matrix, and 33 were differentially expressed on 3-dimensional culture as opposed to plastic. Other than these genes, the patterns of expression were very different in cells grown on SISgel than on Matrigel or even plastic, supporting the hypothesis that growth of bladder cancer cells on normal matrix suppresses some malignant functions. Unique underlying regulatory networks were driving gene expression and could be identified by the approach outlined here.

Proteome-level display by 2-dimensional chromatography of extracellular matrix-dependent modulation of the phenotype of bladder cancer cells.

BACKGROUND: The extracellular matrix can have a profound effect upon the phenotype of cancer cells. Previous work has shown that growth of bladder cancer cells on a matrix derived from normal basement membrane suppresses many malignant features that are displayed when the cells are grown on a matrix that has been modified by malignant tumors. This work was undertaken to investigate proteome-level changes as determined by a new commercially available proteome display involving 2-dimensional chromatography for bladder cancer cells grown on different extracellular matrix preparations that modulate the expression of the malignant phenotype. RESULTS: Depending on the matrix, between 1300 and 2000 distinct peaks were detected by two-dimensional chromatographic fractionation of 2.1-4.4 mg of total cellular protein. The fractions eluting from the reversed-phase fractionation were suitable for mass spectrometric identification following only lyophilization and trypsin digestion and achieved approximately 10-fold higher sensitivity than was obtained with gel-based separations. Abundant proteins that were unique to cells grown on one of the matrices were identified by mass spectrometry. Following concentration, peaks of 0.03 AU provided unambiguous identification of protein components when 10% of the sample was analyzed, whereas peaks of 0.05 AU was approximately the lower limit of detection when the entire sample was separated on a gel and in-gel digestion was used. Although some fractions were homogeneous, others were not, and up to 3 proteins per fraction were identified. Strong evidence for post-translational modification of the unique proteins was noted. All 13 of the unique proteins from cells grown on Matrigel were related to MYC pathway. CONCLUSION: The system provides a viable alternative to 2-dimensional gel electrophoresis for proteomic display of biological systems. The findings suggest the importance of MYC to the malignant phenotype of bladder cancer cells.

The inflammatory and normal transcriptome of mouse bladder detrusor and mucosa.

BACKGROUND: An organ such as the bladder consists of complex, interacting set of tissues and cells. Inflammation has been implicated in every major disease of the bladder, including cancer, interstitial cystitis, and infection. However, scanty is the information about individual detrusor and urothelium transcriptomes in response to inflammation. Here, we used suppression subtractive hybridizations (SSH) to determine bladder tissue- and disease-specific genes and transcriptional regulatory elements (TRE)s. Unique TREs and genes were assembled into putative networks. RESULTS: It was found that the control bladder mucosa presented regulatory elements driving genes such as myosin light chain phosphatase and calponin 1 that influence the smooth muscle phenotype. In the control detrusor network the Pax-3 TRE was significantly over-represented. During development, the Pax-3 transcription factor (TF) maintains progenitor cells in an undifferentiated state whereas, during inflammation, Pax-3 was suppressed and genes involved in neuronal development (synapsin I) were up-regulated. Therefore, during inflammation, an increased maturation of neural progenitor cells in the muscle may underlie detrusor instability. NF-kappaB was specifically over-represented in the inflamed mucosa regulatory network. When the inflamed detrusor was compared to control, two major pathways were found, one encoding synapsin I, a neuron-specific phosphoprotein, and the other an important apoptotic protein, siva. In response to LPS-induced inflammation, the liver X receptor was over-represented in both mucosa and detrusor regulatory networks confirming a role for this nuclear receptor in LPS-induced gene expression. CONCLUSION: A new approach for understanding bladder muscle-urothelium interaction was developed by assembling SSH, real time PCR, and TRE analysis results into regulatory networks. Interestingly, some of the TREs and their downstream transcripts originally involved in organogenesis and oncogenesis were also activated during inflammation. The latter represents an additional link between inflammation and cancer. The regulatory networks represent key targets for development of novel drugs targeting bladder diseases.

Angiogenic factors, bladder neuroplasticity and interstitial cystitis-new pathobiological insights.

Vascular endothelial growth factor (VEGF) is essential for normal embryonic development, and maintenance of adult vascular function. Originally described as a vascular permeability factor, VEGF alters tight cell junctions and contributes to maintenance of bladder permeability. VEGF and its receptors are not only expressed in bladder blood vessels but also in apical cells and intramural ganglia. VEGF receptors are fundamentally altered by inflammation and bladder diseases such as interstitial cystitis (IC). Experimental results indicate that VEGF exerts direct effects on bladder nerve density and function. Regardless of the etiology or initiating cause for IC, it is hypothesized that the urinary bladder responds to injury by increasing the production of VEGF that acts initially as a survival mechanism. However, VEGF also has the capacity to increase vascular permeability leading to glomerulations, edema, and inflammation. Moreover, due to elevated numbers of VEGF receptors in the urothelium, the increased levels of VEGF further increase bladder permeability and establish a vicioCus cycle of disease pathophysiology.