Thrombin modifies growth, proliferation and apoptosis of human colon organoids: a protease-activated receptor 1- and protease-activated receptor 4-dependent mechanism

International audienceExperimental Approach: Crypts were isolated from human colonic resections and cultured for 6 days, forming human colon organoids. Cultured organoids were exposed to 10 and 50 mU·mL−1 of thrombin, in the presence or not of protease‐activated receptor (PAR) antagonists. Organoid morphology, metabolism, proliferation and apoptosis were followed.Key Results: Thrombin favoured organoid maturation leading to a decreased number of immature cystic structures and a concomitant increased number of larger structures releasing cell debris and apoptotic cells. The size of budding structures, metabolic activity and proliferation were significantly reduced in organoid cultures exposed to thrombin, while apoptosis was dramatically increased. Both PAR1 and PAR4 antagonists inhibited apoptosis regardless of thrombin doses. Thrombin‐induced inhibition of proliferation and metabolic activity were reversed by PAR4 antagonist for thrombin's lowest dose and by PAR1 antagonist for thrombin's highest dose.Conclusions and Implications: Overall, our data suggest that the presence of thrombin in the vicinity of human colon epithelial cells favours their maturation at the expense of their regenerative capacities. Our data point to thrombin and its two receptors PAR1 and PAR4 as potential molecular targets for epithelial repair therapies

Modifications fonctionnelles et pharmacologiques de l'appareil urinaire au cours du vieillissement chez le rat

L'OBJECTIF DE CE TRAVAIL A ETE D'EVALUER LES CONSEQUENCES DU VIEILLISSEMENT SUR L'APPAREIL URINAIRE CHEZ LE RAT MALE ET FEMELLE PAR DES APPROCHES CYSTOMANOMETRIQUES, PHARMACOLOGIQUES, HISTOLOGIQUES ET GENOMIQUES. L'ENSEMBLE DE CE TRAVAIL EXPERIMENTAL MONTRE QUE LE VIEILLISSEMENT DE L'APPAREIL URINAIRE CHEZ LE RAT EST CARACTERISE PAR DES MODIFICATIONS DES FONCTIONS VESICALES ET URETRALES, SOUVENT SEMBLABLES CHEZ LE MALE ET LA FEMELLE. EN PARTICULIER, DES INSTABILITES VESICALES ET UN RETARD A LA RELAXATION URETRALE ONT ETE OBSERVES. CES MODIFICATIONS FONCTIONNELLES SONT ASSOCIEES A DES ALTERATIONS HISTOLOGIQUES ET PHARMACOLOGIQUES DIFFERENTES SELON LES NIVEAUX DE L'APPAREIL URINAIRE : LE DOME VESICAL, LA BASE DE VESSIE ET L'URETRE. AU NIVEAU TISSULAIRE, UNE INFILTRATION DE COLLAGENE A ETE SPECIFIQUEMENT LOCALISEE AU NIVEAU DE LA BASE VESICALE, A L'EXCLUSION DU DOME. LES REPONSES FONCTIONNELLES OBTENUES DANS LES DIFFERENTS TISSUS SUGGERENT DES MODIFICATIONS DANS LES NEUROTRANSMISSIONS ADRENERGIQUE, CHOLINERGIQUE ET NANC. LES MODIFICATIONS DE L'EXPRESSION DES GENES AU COURS DU VIEILLISSEMENT VESICAL SUGGERENT QU'UN NOMBRE LIMITE DE PROTEINES, DONT LE FACTEUR DE CROISSANCE NGF, EST IMPLIQUE DANS LES MECANISMES DE LA SENESCENCE. CES RESULTATS MONTRENT QUE LE VIEILLISSEMENT EST RESPONSABLE D'UNE ALTERATION DE LA COORDINATION URETRO-VESICALE ABOUTISSANT A DES MODIFICATIONS SIMILAIRES A CELLES QUI SONT PRESENTES DANS LES PATHOLOGIES OBSTRUCTIVES. LE RAT AGE SEMBLE ETRE UN MODELE EXPERIMENTAL D'INTERET POUR UNE MEILLEURE COMPREHENSION DES MECANISMES DU VIEILLISSEMENT DE L'APPAREIL URINAIRE ET DE SES CONSEQUENCES. AINSI, DE NOUVELLES APPROCHES THERAPEUTIQUES, EN PARTICULIER POUR LE TRAITEMENT DE L'INCONTINENCE URINAIRE, POURRAIENT ETRE PROPOSEES.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocSudocFranceF

Explorations fonctionnelles de la fonction vésicale chez la souris (études urodynamiques, pharmacologiques et génétiques)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Experimental in vivo model to evaluate the impact of Cernitin™ on pain response on induced chronic bladder inflammation

Objective: Inflammation of the urinary bladder may cause burdensome pain also called bladder pain syndrome (BPS). A limitation in understanding BPS pathophysiology is the lack of appropriate preclinical model. Previously published clinical and preclinical studies revealed positive impact of Cernitin™ on pain relief in chronic prostatitis. The objective of this study was to evaluate the effects of Cernitin™ on induced inflammation of the urinary bladder in rats. We also sought to identify biomarkers which might play a role in the management of BPS. Materials and methods: Cystitis was induced by injection of cyclophosphamide (CYP) in female rats. Thereafter, animals were randomly divided into four treatment groups and two control groups. Evaluation of pain scores was assessed by von Frey assay. Expression of pain- and pro-inflammatory biomarkers was determined by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. Results: Treatments with Cernitin™ displayed significant anti-nociceptive effects on CYP-induced visceral pain (p <.01). In contrast, vehicle-treated animals showed high pain score even at the lowest force. Furthermore, results of ELISA showed that Cernitin™-treated animals had significantly reduced levels of COX-2 (T60, p <.01; GBX, p <.05) in bladder tissue homogenate. Immunohistochemical (IHC) staining of bladder tissues showed that Cernitin™-treated animals exhibited less CD45-positive cells, while massive CD45-positive cells infiltration was detected in vehicle-treated animals. IHC also revealed lower SP and PGD2 expression levels in Cernitin™-treated tissues. Conclusions: Cernitin™ components reduced pain score and inflammatory marker COX-2. Our findings suggest a potential therapeutic role for Cernitin™ in the management of BPS

Characterization and Validation of a Chronic Model of Cyclophosphamide-Induced Interstitial Cystitis/Bladder Pain Syndrome in Rats

Interstitial cystitis/Bladder Pain Syndrome (IC/BPS) is a chronic inflammatory disease characterized by visceral pain and voiding symptoms. IC/BPS is still an unsolved enigma with ineffective diagnosis criteria and treatment. A main limitation in IC/BPS understanding is the lack of appropriate preclinical model. Cyclophosphamide (CYP) is commonly used as an experimental model for IC/BPS in rodent. However, the proposed models are very aggressive, contrasting with what occurs in clinic, and often associated with severe toxicity and high mortality rate. In addition, visceral pain, the hallmark symptom of IC/BPS, has been validated in only few of them. In this study, we developed a chronic model of CYP-induced IC/BPS in female rat. In our protocol, no severe weight loss occurred and the survival rate was 100%. In accordance to human pathology, chronic CYP-injected rats developed severe painful behavior whereas only sparse inflammation was observed. Inflammatory response was characterized by bladder edema and focal urothelial damage but absence of massive infiltrate. This chronic model showed persistent symptoms indicative for a central sensitization mechanism. We further demonstrate that CYP-induced chronic visceral pain was significantly reduced by curative treatment with clinically relevant compounds (gabapentin, ibuprofen, and Ialuril ®). We therefore developed and validated a rat model of chronic cystitis that shares strong similarity with human non-ulcerative IC/BPS features without overtly affecting the animal health. This model will thus provide mechanistic insights of the disease and help to evaluate therapeutic agents for IC/BPS

Ketanserin and Naftopidil Enhance the Potentiating Effect of Alpha-Methyl-Serotonin on the Neurally-Induced Contraction of Human Isolated Urinary Bladder Muscle Strips

Purpose The aim of this study was to assess the potential involvement of a specific subtype of 5-hydroxytryptamine (5-HT), 5HT2 receptors in neurally-induced contractions of the human detrusor. Methods Contractile responses to electrical field stimulation (EFS) were examined in human isolated urinary bladder muscle strips. The potentiation of EFS-induced detrusor contraction was examined by adding cumulative concentrations of a 5-HT and 5-HT2 receptor agonist, α-methyl-serotonin (α-Me-5-HT) (1nM–100μM) in the presence or absence of a 5-HT2 antagonist, ketanserin (5-HT2A>5-HT2C) or naftopidil (5-HT2B>5-HT2A) (0.3–3μM). Results 5-HT and α-Me-5-HT potentiated EFS-induced contraction with a maximal effect (Emax) of 37.6% and 38.6%, respectively, and with pEC50 (negative logarithm of the concentration required for a half-maximal response to an agonist) values of 8.3 and 6.8, respectively. Neither ketanserin nor naftopidil at any concentration produced a rightward displacement of the α-Me-5-HT concentration response curve. Instead, the Emax of α-Me-5-HT increased in the presence of ketanserin at 0.3–1μM and in the presence of naftopidil at 1μM to 51% and 56%, respectively, while the Emax in the presence of vehicle alone was 36%. The highest concentration (3μM) of either drug, however, fully reversed the enhancement. Conclusions The potentiating effect of α-Me-5-HT on neurally-induced contraction of human urinary bladder muscle strips was not found to be mediated via any 5-HT2 receptor subtypes. The underlying mechanism for the enhancement of the α-Me-5-HT potentiating effect on detrusor contractility by ketanserin and naftopidil remains unknown; however, our results suggest that these drugs may be useful for treating contractile dysfunction of the detrusor, as manifested in conditions such as underactive bladder

Cellular Senescence in Renal and Urinary Tract Disorders

International audienceCellular senescence is a state of cell cycle arrest induced by repetitive cell mitoses or different stresses, which is implicated in various physiological or pathological processes. The beneficial or adverse effects of senescent cells depend on their transitory or persistent state. Transient senescence has major beneficial roles promoting successful post-injury repair and inhibiting malignant transformation. On the other hand, persistent accumulation of senescent cells has been associated with chronic diseases and age-related illnesses like renal/urinary tract disorders. The deleterious effects of persistent senescent cells have been related, in part, to their senescence-associated secretory phenotype (SASP) characterized by the release of a variety of factors responsible for chronic inflammation, extracellular matrix adverse remodeling, and fibrosis. Recently, an increase in senescent cell burden has been reported in renal, prostate, and bladder disorders. In this review, we will summarize the molecular mechanisms of senescence and their implication in renal and urinary tract diseases. We will also discuss the differential impacts of transient versus persistent status of cellular senescence, as well as the therapeutic potential of senescent cell targeting in these diseases

Impact of Cernitin™ on induced chronic prostatitis in animal model for understanding management of lower urinary tract symptoms

Background: Cernitin™ pollen extracts (brand name Cernilton®) alleviates symptoms related to common lower uro-genital tract disorders in men. The underlying mechanisms are ill-defined but the inflammatory pathway could be one of them. In a previous in vitro study it was shown that Cernitin™ induce a regulatory effect on inflammatory parameters. Methods: In this study, male Sprague Dawley rats were used to validate the effects of Cernitin™ in chronic prostatitis and benign prostatic hyperplasia. Pain was assessed by von Frey assay. Results: Cernitin™ exhibited significant pain relief in the induced prostatitis rat model and was associated with a significant decrease in the intraprostatic level of COX-2 and MCP-1 in the prostatic tissue homogenates. In a parallel study, Cernitin™ treatment led to a significant decrease in prostate weight in rats with testosterone induced BPH. Concurrently, a significant decrease in the percentage of proliferation marker, Ki-67, and androgen receptor expressing cells was observed. Similarly, a low level of cytoplasmic 5α-reductase expression was observed in Cernitin™- and finasteride-treated animals. Conclusion: The current in vivo experiments support the use of Cernitin™ as an anti-inflammatory and symptom reducing agent that could, in part, explain the impact of Cernitin™ on the management of chronic pelvic pain in men

NETUPITANT, A POTENT AND HIGHLY SELECTIVE NK1 RECEPTOR ANTAGONIST, ALLEVIATES ACETIC ACID-INDUCED BLADDER OVERACTIVITY IN ANESTHETIZED GUINEA-PIGS

Introduction. Tachykinins potently contract the isolated urinary bladder from a number of animal species and play an important role in the regulation of the micturition reflex. On the guinea-pig isolated urinary bladder we examined the effects of a new potent and selective NK1 receptor antagonist (netupitant) on the contractions induced by a selective NK1 receptor agonist, SP-methylester (SP-OMe). Moreover, the effects of netupitant and another selective NK1 antagonist (L-733,060) were studied in anesthetized guinea-pigs using two experimental models, the isovolumetric bladder contractions and a model of bladder overactivity induced by intravesical administration of acetic acid (AA). Methods and Results. Detrusor muscle strips were mounted in 5 mL organ baths and isometric contractions to cumulative concentrations of SP-OME were recorded before and after incubation with increasing concentrations of netupitant. In anesthetized female guinea-pigs, reflex bladder activity was examined under isovolumetric conditions with the bladder distended with saline or during cystometry using intravesical infusion of acetic acid (AA). After a 30 min stabilization period, netupitant (0.1-3 mg/kg, i.v.) or L-733,060 (3-10 mg/kg, i.v.) were administered. In the detrusor muscle, netupitant produced a concentration-dependent inhibition (mean pKB = 9.24) of the responses to SP-OMe. Under isovolumetric conditions, netupitant or L-733,060 reduced bladder contraction frequency in a dose-dependent manner, but neither drug changed bladder contraction amplitude. In the AA model, netupitant dose-dependently increased intercontraction interval (ICI) but had no effect on the amplitude of micturition (AM). L-733,060 dose-dependently increased ICI also but this effect was paralleled by a significant reduction of AM. Conclusion. Netupitant decreases the frequency of reflex bladder contractions without altering their amplitude, suggesting that this drug targets the afferent limb of the micturition reflex circuit and therefore may be useful clinically in treating bladder overactivity symptoms

Monoamine Oxidase Inhibitors Prevent Glucose-Dependent Energy Production, Proliferation and Migration of Bladder Carcinoma Cells

International audienceBladder cancer is the 10th most common cancer in the world and has a high risk of recurrence and metastasis. In order to sustain high energetic needs, cancer cells undergo complex metabolic adaptations, such as a switch toward aerobic glycolysis, that can be exploited therapeutically. Reactive oxygen species (ROS) act as key regulators of cancer metabolic reprogramming and tumorigenesis, but the sources of ROS remain unidentified. Monoamine oxidases (MAOs) are mitochondrial enzymes that generate H 2 O 2 during the breakdown of catecholamines and serotonin. These enzymes are particularly important in neurological disorders, but recently, a new link between MAOs and cancer has been uncovered, involving their production of ROS. At present, the putative role of MAOs in bladder cancer has never been evaluated. We observed that human urothelial tumor explants and the bladder cancer cell line AY27 expressed both MAO-A and MAO-B isoforms. Selective inhibition of MAO-A or MAO-B limited mitochondrial ROS accumulation, cell cycle progression and proliferation of bladder cancer cells, while only MAO-A inhibition prevented cell motility. To test whether ROS contributed to MAO-induced tumorigenesis, we used a mutated form of MAO-A which was unable to produce H 2 O 2. Adenoviral transduction of the WT MAO-A stimulated the proliferation and migration of AY27 cells while the Lys305Met MAO-A mutant was inactive. This was consistent with the fact that the antioxidant Trolox strongly impaired proliferation and cell cycle progression. Most interestingly, AY27 cells were highly dependent on glucose metabolism to sustain their growth, and MAO inhibitors potently reduced glycolysis and oxidative phosphorylation, due to pyruvate depletion. Accordingly, MAO inhibitors decreased the expression of proteins involved in glucose transport (GLUT1) and transformation (HK2). In conclusion, urothelial cancer cells are characterized by a metabolic shift toward glucose-dependent metabolism, which is important for cell growth and is under the regulation of MAO-dependent oxidative stress