RESUMO
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.
Assuntos
Neurônios Motores/fisiologia , Plasticidade Neuronal/fisiologia , Nervos Periféricos/fisiologia , Células Receptoras Sensoriais/fisiologia , Bexiga Urinária/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Vísceras/fisiologia , Administração Intravesical , Animais , Neurônios Colinérgicos/metabolismo , Neurônios Colinérgicos/fisiologia , Feminino , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Motores/metabolismo , Nervos Periféricos/metabolismo , Células Receptoras Sensoriais/metabolismo , Canais de Cátion TRPV/metabolismo , Bexiga Urinária/inervação , Bexiga Urinária/metabolismo , Micção/fisiologia , Vísceras/inervação , Vísceras/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
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.
Assuntos
Vacina BCG/farmacologia , Inflamação/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Vacina BCG/imunologia , Calcitonina/imunologia , Calcitonina/metabolismo , Feminino , Inflamação/induzido quimicamente , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/imunologia , Neuropilina-1/imunologia , Neuropilina-1/metabolismo , Neuropilina-2/imunologia , Neuropilina-2/metabolismo , Neuropilinas/efeitos dos fármacos , Neuropilinas/imunologia , Neuropilinas/metabolismo , Precursores de Proteínas/imunologia , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/farmacologia , Semaforinas/imunologia , Semaforinas/metabolismo , Transdução de Sinais , Substância P/imunologia , Substância P/metabolismo , Canais de Cátion TRPV/imunologia , Canais de Cátion TRPV/metabolismo , Ubiquitina Tiolesterase/imunologia , Ubiquitina Tiolesterase/metabolismo , Bexiga Urinária/imunologia , Bexiga Urinária/patologia , Urotélio/efeitos dos fármacos , Urotélio/imunologia , Urotélio/metabolismoRESUMO
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.
Assuntos
Cistite/fisiopatologia , Neuropilina-1/fisiologia , Neuropilinas/fisiologia , Receptores de Fatores de Crescimento do Endotélio Vascular/fisiologia , Transdução de Sinais/fisiologia , Animais , Vacina BCG , Movimento Celular/imunologia , Cistite/induzido quimicamente , Feminino , Humanos , Linfangiogênese/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/fisiopatologia , Neuropilina-1/imunologia , Bexiga Urinária/irrigação sanguínea , Bexiga Urinária/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
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.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Boswellia , Óleos Voláteis/uso terapêutico , Extratos Vegetais/uso terapêutico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética , Urotélio/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Resinas Vegetais , Fatores de Transcrição , Urotélio/citologiaRESUMO
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.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Mycobacterium bovis/imunologia , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Bexiga Urinária/imunologia , Urotélio/imunologia , Animais , Cistite Intersticial/imunologia , Cistite Intersticial/terapia , Feminino , Regulação da Expressão Gênica/imunologia , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/imunologia , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/terapiaRESUMO
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.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Cistite/metabolismo , Redes Reguladoras de Genes , Inflamação/metabolismo , Receptores Ativados por Proteinase/metabolismo , Bexiga Urinária/metabolismo , Animais , Feminino , Mastócitos/citologia , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL/metabolismo , Camundongos Mutantes , Mucosa/citologia , Mucosa/metabolismo , Receptores Ativados por Proteinase/isolamento & purificaçãoRESUMO
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.
Assuntos
Vacina BCG/administração & dosagem , Cistite/induzido quimicamente , Cistite/urina , Citocinas/metabolismo , Interleucina-17/urina , Bexiga Urinária/efeitos dos fármacos , Administração Intravesical , Animais , Imunoprecipitação da Cromatina , Cistite/patologia , Citocinas/genética , Citocinas/urina , Modelos Animais de Doenças , Feminino , Expressão Gênica/efeitos dos fármacos , Granuloma/induzido quimicamente , Granuloma/patologia , Granuloma/urina , Imuno-Histoquímica , Interleucina-17/genética , Interleucina-17/metabolismo , Lipopolissacarídeos/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/patologia , Neutrófilos/ultraestrutura , Fator de Necrose Tumoral alfa/administração & dosagem , Regulação para Cima , Bexiga Urinária/imunologia , Bexiga Urinária/ultraestruturaRESUMO
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.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma in Situ/diagnóstico , Carcinoma in Situ/patologia , Vasos Linfáticos/diagnóstico por imagem , Vasos Linfáticos/patologia , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/patologia , Animais , Carcinoma in Situ/imunologia , Modelos Animais de Doenças , Glicoproteínas/análise , Glicoproteínas/imunologia , Imuno-Histoquímica , Linfangiogênese , Vasos Linfáticos/metabolismo , Linfografia/métodos , Imageamento por Ressonância Magnética , Proteínas de Membrana/análise , Proteínas de Membrana/imunologia , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Espectroscopia de Luz Próxima ao Infravermelho , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/imunologia , Uroplaquina IIRESUMO
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.
Assuntos
Apresentação de Antígeno/genética , Vacina BCG/farmacologia , GTP Fosfo-Hidrolases/biossíntese , Regulação Neoplásica da Expressão Gênica/imunologia , Antígenos HLA/biossíntese , Antígenos de Histocompatibilidade Classe II/biossíntese , Proteínas de Membrana/biossíntese , Bexiga Urinária/efeitos dos fármacos , Urotélio/efeitos dos fármacos , Animais , Apresentação de Antígeno/imunologia , Vacina BCG/administração & dosagem , Northern Blotting , Imunoprecipitação da Cromatina , Feminino , GTP Fosfo-Hidrolases/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antígenos H-2/biossíntese , Antígenos H-2/genética , Antígenos de Histocompatibilidade Classe II/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Técnica de Subtração , Bexiga Urinária/metabolismoRESUMO
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.
Assuntos
Cistite/genética , Cistite/metabolismo , Regulação da Expressão Gênica , Receptor PAR-1/metabolismo , Animais , Antígenos/imunologia , Cálcio/metabolismo , Cromatina/metabolismo , Cistite/induzido quimicamente , Cistite/imunologia , Feminino , Expressão Gênica , Genoma , Imunoprecipitação , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Fosfolipases A/metabolismo , Reação em Cadeia da Polimerase/métodos , Receptor PAR-1/deficiência , Frações Subcelulares/metabolismo , Substância P , Bexiga Urinária/metabolismoRESUMO
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.
Assuntos
Cistite/metabolismo , Receptor PAR-1/metabolismo , Animais , Antígenos/imunologia , Linhagem Celular , Cistite/induzido quimicamente , Cistite/imunologia , Cistite/patologia , Edema/induzido quimicamente , Granulócitos/patologia , Humanos , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor PAR-1/agonistas , Receptor PAR-1/deficiência , Receptor PAR-2/efeitos dos fármacos , Receptor PAR-2/metabolismo , Receptores Ativados por Proteinase/metabolismo , Substância P , Bexiga Urinária/metabolismo , Urotélio/citologia , Urotélio/metabolismoRESUMO
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.
Assuntos
Cistite/genética , Cistite/imunologia , Receptores da Neurocinina-1/genética , Receptores da Neurocinina-1/metabolismo , Elementos Reguladores de Transcrição/genética , Taquicininas/imunologia , Bexiga Urinária/imunologia , Animais , Feminino , Fatores Imunológicos/genética , Fatores Imunológicos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteoma/genética , Proteoma/imunologia , Taquicininas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/imunologiaRESUMO
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.
Assuntos
Cistite/genética , Cistite/imunologia , Genômica , Transcrição Gênica , Bexiga Urinária/fisiologia , Animais , Cistite/fisiopatologia , DNA Complementar , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Biblioteca Gênica , Hibridização Genética , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Urotélio/fisiologiaRESUMO
Inflammation is an inherent response of the organism that permits its survival despite constant environmental challenges. The process normally leads to recovery from injury and to healing. However, if targeted destruction and assisted repair are not properly phased, chronic inflammation can result in persistent tissue damage. To better understand the inflammatory process, we recently introduced a profiling methodology to identify common genes involved in bladder inflammation. The method represents a complementation to the classic quantification of inflammation and provides information regarding the early, intermediate, and late events in gene regulation. However, gene profiling fails to describe the molecular pathways and their interconnections involved in the particular inflammatory response. The present work introduces a new statistical technique for inferring functional interconnections between inflammatory pathways underlying classic models of bladder inflammation and permits the modeling of the inflammatory network. This new statistical method is based on variants of cluster analysis, Boolean networking, differential equations, Bayesian networking, and partial correlation. By applying partial correlation analysis, we developed mosaics of gene expression that permitted a global visualization of common and unique pathways elicited by different stimuli. The significance of these processes was tested from both biological and statistical viewpoints. We propose that connective mosaic may represent the necessary simplification step to visualize cDNA array results.
Assuntos
Cistite/genética , Perfilação da Expressão Gênica/métodos , Animais , Análise por Conglomerados , Cistite/induzido quimicamente , Dinitrofenóis/imunologia , Dinitrofenóis/farmacologia , Feminino , Perfilação da Expressão Gênica/estatística & dados numéricos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Variação Genética , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência com Séries de Oligonucleotídeos/estatística & dados numéricos , Ovalbumina/imunologia , Ovalbumina/farmacologia , Albumina Sérica/imunologia , Albumina Sérica/farmacologia , Substância P/efeitos adversos , Substância P/farmacologiaRESUMO
Neurokinin 1 (NK(1)) receptors play a fundamental role in neurogenic inflammation. We sought to determine the mechanisms downstream from NK(1) receptor (NK(1)R) activation using cDNA arrays and a novel statistical method to analyze gene expression. We used female NK(1)R(-/-) and wild-type (WT) mice that were sensitized actively by intraperitoneal injections of dinitrophenol 4 (DNP(4))-human serum albumin. Cystitis was induced by intravesical instillation of antigen of DNP(4)-ovalbumin, and control mice were challenged with saline. At 1, 4, and 24 h after instillation, bladders were removed for 1) RNA extraction (n = 3), 2) replicate of RNA extraction (n = 3), and 3) morphological analysis (n = 6). For cDNA array experiments, three bladders from each group were homogenized, and total RNA was obtained. DNase-treated RNA was reverse-transcribed to cDNA, labeled with [alpha-(32)P]dATP and hybridized to Atlas Mouse 1.2 Arrays (Clontech). After calculating the mean and SD for background spots, each experimental value was assigned a normalized score S using the formula S' = (S - Av)/SD, where S' is the original pixel value, and Av and SD are the mean and standard deviation of background spots, respectively. Only genes that expressed 3 SD values above background were used. Hypervariable genes were sorted by cluster analysis. Matrices of correlation coefficients were calculated and represented in a connectivity mosaic. As results, we found that in WT mice the most prominent gene cluster had neprilysin in a central position and positively correlated to a group of activator protein-1 (AP-1)-responsive genes, including laminin-alpha3, tissue plasminogen activator 11, fos-B, and TNF-beta. In WT mice, antigen-induced bladder inflammation led to a downregulation in neprilysin expression. In contrast, NK(1)R(-/-) mice failed to mount an inflammatory reaction and presented neprilysin negatively correlated with the same genes described in WT. In conclusion, this work indicates an overriding participation of NK(1)R and neprilysin in bladder inflammation, provides a working model for the involvement of AP-1 transcription factor, and evokes testable hypotheses regarding the role of NK(1)R and neprilysin in inflammation.
Assuntos
Neprilisina/fisiologia , Receptores da Neurocinina-1/fisiologia , Bexiga Urinária/metabolismo , Animais , Análise por Conglomerados , Cistite/genética , Regulação da Expressão Gênica , Genótipo , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência com Séries de Oligonucleotídeos/normas , Receptores da Neurocinina-1/genética , Reprodutibilidade dos Testes , Bexiga Urinária/patologiaRESUMO
Inflammation underlies all major bladder pathologies including malignancy and represents a defense reaction to injury caused by physical damage, chemical substances, micro-organisms or other agents. During acute inflammation, activation of specific molecular pathways leads to an increased expression of selected genes whose products attack the insult, but ultimately should protect the tissue from the noxious stimulus. However, once the stimulus ceases, gene-expression should return to basal levels to avoid tissue damage, fibrosis, loss of function, and chronic inflammation. If this down-regulation does not occur, tissue fibrosis occurs as a serious complication of chronic inflammation. Although sensory nerve and most cells products are known to be key parts of the inflammatory puzzle, other key molecules are constantly being described that have a role in bladder inflammation. Therefore, as the database describing the repertoire of inflammatory mediators implicated in bladder inflammation increases, the central mechanisms by which injury can induce inflammation, cell damage, and repair often becomes less rather than more clear. To make sense of the vast knowledge of the genes involved in the inflammatory response may require analysis of the patterns of change and the elucidation of gene networks far more than definition of additional members of inflammatory cascades. This review discuss the appropriate use of microarray technology, which promises to solve both of these problems as well as identifying key molecules and mechanisms involved in the transition between acute and chronic inflammation.
Assuntos
Perfilação da Expressão Gênica , Inflamação/patologia , Doenças da Bexiga Urinária/genética , Animais , Previsões , Genoma , Genômica , Humanos , Modelos Biológicos , Neprilisina/metabolismo , ProteômicaRESUMO
Interstitial cystitis (IC) is a chronic and painful bladder syndrome of unknown cause with no reliable biological marker or effective therapy. Vascular endothelial growth factor (VEGF), which plays a key role in bladder inflammation, is closely associated with the vascular alterations observed in patients with IC. However, our recent findings of VEGF receptors (VEGF-Rs) and VEGF coreceptors on nonendothelial cells in human and mouse urothelium suggest that additional VEGF targets and functions are possible in IC bladders. We report here that VEGF-Rs and coreceptors (neuropilins; NRP) are strongly expressed in both the human bladder urothelium and in the human bladder cancer cell line (J82) and that the expression of NRP2 and VEGF-R1 is significantly downregulated in IC compared with control subjects. In addition, treatment of J82 cells with bacillus Calmette-Guérin (BCG), a novel treatment strategy for IC, upregulates the messages for NRPs and VEGF-Rs. Furthermore, intravesical instillation of an internalizable VEGF fluorescent tracer (scVEGF/Cy5.5) into mouse urinary bladders results in a marked ligand accumulation in the urothelium and bladder parenchyma, indicating that urothelial VEGF-Rs are functionally active and capable of ligand interaction and internalization. Our results suggest that the VEGF pathway is altered in IC, that urinary VEGF may gain access to the bladder wall via these receptors, and that BCG treatment may replenish the missing VEGF-Rs/NRP receptors. Together, these results suggest that levels of NRPs, VEGF-Rs, and VEGF are new putative markers for the diagnosis of IC and that modulating these receptors can be exploited as therapeutic strategies.
Assuntos
Cistite Intersticial/fisiopatologia , Neuropilinas/fisiologia , Receptores de Fatores de Crescimento do Endotélio Vascular/fisiologia , Animais , Linhagem Celular , Cistite Intersticial/genética , Feminino , Humanos , Inflamação/fisiopatologia , Masculino , Camundongos , Reação em Cadeia da Polimerase , Bexiga Urinária/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Recent evidence supports a role for vascular endothelium growth factor (VEGF) signaling in bladder inflammation. However, it is not clear what bladder cells are targeted by VEGF. Therefore, we determined the nature of cells responding to VEGF in normal and inflamed bladders by tagging such cells in vivo with a targeted fluorescent tracer, scVEGF/Cy, an engineered single-chain VEGF labeled with Cy5.5 dye, which identifies cells with accessible and functionally active VEGF receptors. Inflammation was induced by intravesical instillation of PAR-activating peptides or BCG. In vivo NIRF imaging with intravenously injected scVEGF/Cy revealed accumulation of the tracer in the control mouse bladder and established that inflammation increased the steady-state levels of tracer uptake. Ex vivo colocalization of Cy5.5 dye revealed that in normal and at a higher level in inflamed bladder, accumulation of scVEGF/Cy occurs in both urothelial and ganglial cells, expressing VEGF receptors VEGFR-1 and VEGFR-2, as well as VEGF coreceptors neuropilins (NRP) NRP1 and NRP2. PCR results indicate that the messages for VEGF-Rs and NRPs are present in the bladder mucosa and ChIP/QPCR analysis indicated that inflammation induced upregulation of genes encoding VEGFRs and NRPs. Our results strongly suggest new and blossoming VEGF-driven processes in bladder urothelial cells and ganglia in the course of inflammation. We expect that molecular imaging of the VEGF pathway in the urinary tract by receptor-mediated cell tagging in vivo will be useful for clinical diagnosis and therapeutic monitoring, and will help to accelerate the development of bladder-targeting drugs and treatments.
Assuntos
Cistite/metabolismo , Neuropilinas/biossíntese , Receptores de Fatores de Crescimento do Endotélio Vascular/biossíntese , Bexiga Urinária/metabolismo , Adulto , Idoso , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Neurônios/metabolismo , Neuropilina-1/biossíntese , Neuropilina-2/biossíntese , Espectroscopia de Luz Próxima ao Infravermelho , Bexiga Urinária/citologia , Urotélio/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossínteseRESUMO
A spatial association between mast cells and nerves has been described in both the gastrointestinal and genitourinary tracts. However, the factors that influence the anatomic relationship between mast cells and nerves have not been completely defined. It has been suggested that the high-affinity receptor for substance P [neurokinin-1 (NK1)] might modulate this interaction. We therefore assessed mast cell-nerve relationships in tissues isolated from wild-type and NK1 receptor knockout (NK1-/-) mice. We now report that, in the complete absence of NK1 receptor expression, there is a significant increase in the number of mast cells without a change in the anatomic relationship between mast cell and nerves in stomach and bladder tissues at the light microscopic level. We next determined whether transplanted mast cells would maintain their spatial distribution, number, and contact with nerve elements. For this purpose, mast cell-deficient Kit(W)/Kit(W-v) mice were reconstituted with wild-type or NK1-/- bone marrow. No differences in mast cell-nerve contact were observed. These results suggest that NK1 receptor expression is important in the regulation of the number of mast cells but is not important in the interaction between mast cells and nerves. Furthermore, the interaction between mast cells and nerves is not mediated through NK1 receptor expression on the mast cell. Further studies are needed to determine the molecular pathway involved in mast cell migration and interaction with nerve elements, but the model of reconstitution of Kit(W)/Kit(W-v) mice with mast cells derived from different genetically engineered mice is a useful approach to further explore these mechanisms.
Assuntos
Mastócitos/fisiologia , Neurônios/fisiologia , Receptores da Neurocinina-1/metabolismo , Animais , Transplante de Medula Óssea , Contagem de Células , Feminino , Expressão Gênica , Camundongos , Camundongos Knockout , Estômago/anatomia & histologia , Estômago/inervação , Bexiga Urinária/anatomia & histologia , Bexiga Urinária/inervaçãoRESUMO
Inflammatory bladder disorders such as interstitial cystitis (IC) deserve attention since a major problem of the disease is diagnosis. IC affects millions of women and is characterized by severe pain, increased frequency of micturition, and chronic inflammation. Characterizing the molecular fingerprint (gene profile) of IC will help elucidate the mechanisms involved and suggest further approaches for therapeutic intervention. Therefore, in the present study we used established animal models of cystitis to determine the time course of bladder inflammatory responses to antigen, Escherichia coli lipopolysaccharide (LPS), and substance P (SP) by morphological analysis and cDNA microarrays. The specific aim of the present study was to compare bladder inflammatory responses to antigen, LPS, and SP by morphological analysis and cDNA microarray profiling to determine whether bladder responses to inflammation elicit a specific universal gene expression response regardless of the stimulating agent. During acute bladder inflammation, there was a predominant infiltrate of polymorphonuclear neutrophils into the bladder. Time-course studies identified early, intermediate, and late genes that were commonly up-regulated by all three stimuli. These genes included: phosphodiesterase 1C, cAMP-dependent protein kinase, iNOS, beta-NGF, proenkephalin B and orphanin, corticotrophin-releasing factor (CRF) R, estrogen R, PAI2, and protease inhibitor 17, NFkB p105, c-fos, fos-B, basic transcription factors, and cytoskeleton and motility proteins. Another cluster indicated genes that were commonly down-regulated by all three stimuli and included HSF2, NF-kappa B p65, ICE, IGF-II and FGF-7, MMP2, MMP14, and presenilin 2. Furthermore, we determined gene profiles that identify the transition between acute and chronic inflammation. During chronic inflammation, the urinary bladder presented a predominance of monocyte/macrophage infiltrate and a concomitant increase in the expression of the following genes: 5-HT 1c, 5-HTR7, beta 2 adrenergic receptor, c-Fgr, collagen 10 alpha 1, mast cell factor, melanocyte-specific gene 2, neural cell adhesion molecule 2, potassium inwardly-rectifying channel, prostaglandin F receptor, and RXR-beta cis-11-retinoic acid receptor. We conclude that microarray analysis of genes expressed in the bladder during experimental inflammation may be predictive of outcome. Further characterization of the inflammation-induced gene expression profiles obtained here may identify novel biomarkers and shed light into the etiology of cystitis.