Giorgia Colciago

Expression of Fatty Acid Amide Hydrolase (FAAH) in Human, Mouse, and Rat Urinary Bladder and Effects of FAAH Inhibition on Bladder Function in Awake Rats.

BACKGROUND Cannabinoid receptor (CB)-mediated functions may be involved in the regulation of bladder function, but information on endocannabinoid signals during micturition is scarce. OBJECTIVE Investigate the expression of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) in human, rat, and mouse bladders and study the effects of inhibition of FAAH during urodynamics in awake rats. DESIGN, SETTING, AND PARTICIPANTS Bladder tissue from humans, mice, and rats was used for measurements. Female Sprague-Dawley rats were administered the FAAH inhibitor oleoyl ethyl amide (OEtA) or vehicle intravenously (IV) or intravesically (IVES) with or without rimonabant (CB1 antagonist) or SR144528 (CB2 antagonist). MEASUREMENTS Real-time transcriptase-polymerase chain reaction, Western blot, immunohistochemistry, and cystometry in awake rats. RESULTS AND LIMITATIONS Messenger RNA and protein for FAAH was expressed in the mucosa of human, mouse, and rat urinary bladders. Immunoreactivities for FAAH and CB2 were codistributed in rat and human urothelium. IV OEtA (0.3mg/kg) to rats increased intercontraction intervals (ICIs), micturition volume (MV), bladder capacity (BC), and threshold pressure (TP) by 17±1%, 16±1%, 17±1%, and 19±5%, respectively (all p<0.05 vs baseline). IVES OEtA (1 and 10mg/l) in rats dose-dependently increased (p<0.05 vs baseline) ICI (19±2% and 35±5%), MV (15±3% and 32±4%), BC (16±2% and 34±4%), and TP (15±1%, 21±3%). SR144528 (IVES 5mg/l) abolished all effects of OEtA, whereas rimonabant only counteracted effects of OEtA on TP. CONCLUSIONS Bladder mucosa of all species expressed FAAH. Rat and human urothelium coexpressed FAAH and CB2. The FAAH inhibitor OEtA altered urodynamic parameters that reflect sensory functions of micturition in rats. Suggesting a role for the endocannabinoid system in bladder mechanoafferent functions of rats, effects of IVES OEtA were abolished by an IVES CB2 antagonist and partly counteracted by an IVES CB1 antagonist.

Mesenchymal stem cells expressing therapeutic genes induce autochthonous prostate tumour regression

Mesenchymal stem cells (MSC) as vehicles of therapeutic genes represent a unique tool to activate drugs within a neoplastic mass due to their property to home and engraft into tumours. In particular, MSC expressing the cytosine deaminase::uracil phosphoribosyltransferase (CD-MSC) have been previously demonstrated to inhibit growth of subcutaneous prostate cancer xenografts thanks to their ability to convert the non-toxic 5-fluorocytosine into the antineoplastic 5-fluorouracil. Since both the immune system and the tumour microenvironment play a crucial role in directing cancer progression, in order to advance towards clinical applications, we tested the therapeutic potential of this approach on animal models that develop autochthonous prostate cancer and preserve an intact immune system. As cell vectors, we employed adipose-tissue and bone-marrow MSC. CD-MSC toxicity on murine prostate cancer cells and tumour tropism were verified in vitro and ex-vivo before starting the preclinical studies. Magnetic Resonance Imaging was utilised to follow orthotopic tumour progression. We demonstrated that intravenous injections of CD-MSC cells, followed by intraperitoneal administration of 5-fluorocytosine, caused tumour regression in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model, which develops aggressive and spontaneous prostate cancer. These results add new insights to the therapeutic potential of specifically engineered MSC in prostate cancer disease.

Bladder cancer cell growth and motility implicate cannabinoid 2 receptor-mediated modifications of sphingolipids metabolism

The inhibitory effects demonstrated by activation of cannabinoid receptors (CB) on cancer proliferation and migration may also play critical roles in controlling bladder cancer (BC). CB expression on human normal and BC specimens was tested by immunohistochemistry. Human BC cells RT4 and RT112 were challenged with CB agonists and assessed for proliferation, apoptosis, and motility. Cellular sphingolipids (SL) constitution and metabolism were evaluated after metabolic labelling. CB1-2 were detected in BC specimens, but only CB2 was more expressed in the tumour. Both cell lines expressed similar CB2. Exposure to CB2 agonists inhibited BC growth, down-modulated Akt, induced caspase 3-activation and modified SL metabolism. Baseline SL analysis in cell lines showed differences linked to unique migratory behaviours and cytoskeletal re-arrangements. CB2 activation changed the SL composition of more aggressive RT112 cells by reducing (p < 0.01) Gb3 ganglioside (−50 ± 3%) and sphingosine 1-phosphate (S1P, −40 ± 4%), which ended up to reduction in cell motility (−46 ± 5%) with inhibition of p-SRC. CB2-selective antagonists, gene silencing and an inhibitor of SL biosynthesis partially prevented CB2 agonist-induced effects on cell viability and motility. CB2 activation led to ceramide-mediated BC cell apoptosis independently of SL constitutive composition, which instead was modulated by CB2 agonists to reduce cell motility.

DFL23448, A Novel Transient Receptor Potential Melastin 8–Selective Ion Channel Antagonist, Modifies Bladder Function and Reduces Bladder Overactivity in Awake Rats

The transient receptor potential melastin 8 ion channel (TRPM8) is implicated in bladder sensing but limited information on TRPM8 antagonists in bladder overactivity is available. This study characterizes a new TRPM8-selective antagonist (DFL23448 [5-(2-ethyl-2H-tetrazol-5-yl)-2-(3-fluorophenyl)-1,3-thiazol-4-ol]) and evaluates it in cold-induced behavioral tests and tests on bladder function and experimental bladder overactivity in vivo in rats. DFL23448 displayed IC50 values of 10 and 21 nM in hTRPM8 human embryonic kidney 293 cells activated by Cooling Agent 10 or cold, but it had limited activity (IC50 > 10 μM) at transient receptor potential vanilloids TRPV1, TRPA1, or TRPV4 or at various G protein–coupled receptors. In rats, DFL23448 administered intravenously or orally had a half-life of 37 minutes or 4.9 hours, respectively. DLF23448 (10 mg/kg i.v.) reduced icilin-induced “wet dog–like” shakes in rats. Intravesical DFL23448 (10 mg/l), but not vehicle, increased micturition intervals, micturition volume, and bladder capacity. During bladder overactivity by intravesical prostaglandin E2 (PGE2), vehicle controls exhibited reductions in micturition intervals, micturition volumes, and bladder capacity by 37%–39%, whereas the same parameters only decreased by 12%–15% (P < 0.05–0.01 versus vehicle) in DFL23448-treated rats. In vehicle-treated rats, but not in DFL23448-treated rats, intravesical PGE2 increased bladder pressures. Intravenous DFL23448 at 10 mg/kg, but not 1 mg/kg DFL23448 or vehicle, increased micturition intervals, micturition volumes, and bladder capacity. During bladder overactivity by intravesical PGE2, micturition intervals, micturition volumes, and bladder capacity decreased in vehicle– and 1 mg/kg DFL23448–treated rats, but not in 10 mg/kg DFL23448–treated rats. Bladder pressures increased less in rats treated with DFL23448 10 mg/kg than in vehicle– or 1 mg/kg DFL23448–treated rats. DFL23448 (10 mg/kg i.v.), but not vehicle, prevented cold stress–induced bladder overactivity. Our results support a role for bladder TRPM8-mediated signals in experimental bladder overactivity.

Ouabain Contributes to Kidney Damage in a Rat Model of Renal Ischemia-Reperfusion Injury

Warm renal ischemia performed during partial nephrectomy has been found to be associated with kidney disease. Since endogenous ouabain (EO) is a neuro-endocrine hormone involved in renal damage, we evaluated the role of EO in renal ischemia-reperfusion injury (IRI). We measured plasma and renal EO variations and markers of glomerular and tubular damage (nephrin, KIM-1, Kidney-Injury-Molecule-1, α1 Na-K ATPase) and the protective effect of the ouabain inhibitor, rostafuroxin. We studied five groups of rats: (1) normal; (2) infused for eight weeks with ouabain (30 µg/kg/day, OHR) or (3) saline; (4) ouabain; or (5) saline-infused rats orally treated with 100 µg/kg/day rostafuroxin for four weeks. In group 1, 2–3 h after IRI, EO increased in ischemic kidneys while decreased in plasma. Nephrin progressively decreased and KIM-1 mRNA increased starting from 24 h. Ouabain infusion (group 2) increased blood pressure (from 111.7 to 153.4 mmHg) and ouabain levels in plasma and kidneys. In OHR ischemic kidneys at 120 h from IRI, nephrin, and KIM-1 changes were greater than those detected in the controls infused with saline (group 3). All these changes were blunted by rostafuroxin treatment (groups 4 and 5). These findings support the role of EO in IRI and suggest that rostafuroxin pre-treatment of patients before partial nephrectomy with warm ischemia may reduce IRI, particularly in those with high EO.

MP43-10 THE ENDOCANNABINOID SYSTEM IN MALE REPRODUCTIVE FUNCTION: EXPRESSION AND LOCALIZATION OF FATTY ACID AMIDE HYDROLASE (FAAH) IN THE SEMINAL VESICLES AND VAS DEFERENS

INTRODUCTION AND OBJECTIVES: The endocannabinoid system (ECS), comprising the cannabinoid receptors (CBR), their ligands, and enzymes controlling the turn-over of (endo)cannabinoids (anandamide, tetrahydrocannabinol 1⁄4 THC) , has been suggested to be involved in male reproductive function. As information is scares on the expression of the ECS in human male reproductive tissues, the present study aimed to investigate in the human seminal vesicles (SV) and Vas deferens (VD) by means of molecular biology (RT-PCR) and immunohistochemistry (IHC)/immunofluorescence the expression and distribution of Fatty Acid Amide Hydrolase (FAAH, isoforms 1 and 2, an enzyme known to degrade/hydrolyze cannabinoids, such as anandamide). METHODS: Macroscopically normal specimens of VS and VD were obtained from 5 male subjects who underwent pelvic surgery for urological malignancies (carcinoma of the prostate/urinary bladder). The tissue was processed for molecular biology analysis (Polymerase Chain Reaction 1⁄4 RT-PCR), Western blotting and immunohistochemistry. Cryostat sections were exposed to specific antibodies directed against FAAH1 and FAAH2, visualization was commenced by means of immunofluorescence. RESULTS: All specimens expressed PCR products corresponding to FAAH1 (260 bp) and FAAH2 (387 bp). Western blot analysis displayed distinct lanes at positions of the expected molecular weight of the FAAH1 (63 kDa) and FAAH2 protein (68 kDa). IHC revealed cytosolic staining for FAAH1 and FAAH2 in cuboidal cells of the pseudo-stratified epithelial multilayer. No immunoreactivity was detected in the smooth musculature or nerve fibers transversing the tissue. CONCLUSIONS: Genes encoding for FAAH1 and FAAH2 are highly expressed in the human SV and VD. Considering the localization of the enzymes (as demonstrated by means of IHC), the ECS may be involved in epithelial homeostasis/secretory function rather than autonomic mechano-afferent signaling (control of smooth muscle).

Reply to Manish Garg, Apul Goel and Jai Prakash's Letter to the Editor re: Renzo Colombo, Lorenzo Rocchini, Nazareno Suardi, et al. Neoadjuvant Short-term Intensive Intravesical Mitomycin C Regimen Compared with Weekly Schedule for Low-grade Recurrent Non–muscle-invasive Bladder Cancer: Preliminary Results of a Randomised Phase 2 Study. Eur Urol 2012;62:797–802

We appreciated the comments in the letter from Garg and colleagues [1] concerning our paper [2]. Garg et al. mainly concur and emphasize the need for clinical trials specifically designed to overcome the general approach of administering neoadjuvant intravesical chemotherapy based on empirical findings rather than on a commonly recognized scientific rationale. In their letter [1], the authors state that although the ideal total number of mitomycin C (MMC) instillations has yet to be defined, the setting used in our trial (six instillations) might not be optimal. Garg et al. cite the 1975 article by Mishina et al. [3], in which an intensive schedule similar to ours (three times weekly) [2] was used but included a total of 20 instillations over 7 wk. Garg et al. claim that better results in terms of tumor response would have been achieved in our study had we adhered to the schedule used by Mishina et al. In considering the trial by Mishina et al. [3], it should be noted that the trial was not randomized and that toxicity was not measured with a validated questionnaire. More important, the dose/concentration of MMC was reduced (20 mg/20 ml) when compared with that used in our study [2]. The total number of instillations used by Mishina et al. was determined only on an empirical basis (eg, why 20?) and, unlike findings reported by Whelan [4], only 44% and 76% tumor complete response and overall tumor response, respectively, could be documented. Consequently, it seems like perhaps the dose/concentration of the drug might have an impact on the oncologic outcome more consistently than the overall number of administrations. Likewise, Garg et al. [1] state that adapting our neoadjuvant chemotherapy schedule [2] to the optimized method for MMC administration proposed by Au et al. [5] (including overnight dehydration, repeat doses of oral bicarbonate, complete emptying of the bladder, and changing position on the bed) would have improved