Roberta Buono

The low-affinity receptor for neurotrophins p75NTR plays a key role for satellite cell function in muscle repair acting via RhoA.

Regeneration of muscle fibers, lost during pathological muscle degeneration or after injuries, is mediated by the production of new myofibres. This process, sustained by the resident stem cells of the muscle, the satellite cells, is finely regulated by local cues, in particular by cytokines and growth factors. Evidence in the literature suggests that nerve growth factor (NGF) is involved in muscle fiber regeneration; however, its role and mechanism of action were unclear. We have investigated this issue in in vivo mouse models of muscle regeneration and in primary myogenic cells. Our results demonstrate that NGF acts through its low-affinity receptor p75(NTR) in a developmentally regulated signaling pathway necessary to myogenic differentiation and muscle repair in vivo. We also demonstrate that this action of NGF is mediated by the down-regulation of RhoA-GTP signaling in myogenic cells.

Necdin is expressed in cachectic skeletal muscle to protect fibers from tumor-induced wasting

Skeletal muscles of subjects with advanced cancer undergo progressive wasting, referred to as cachexia. Cachexia is an important area for medical research because strategies proposed until now have yielded little benefit. We have recently identified necdin as a key player in fetal and postnatal physiological myogenesis and in muscle regeneration. Here we show that necdin is selectively expressed in muscles of cachetic mice and prove that its expression is causally linked to a protective response of the tissue against tumor-induced wasting, inhibition of myogenic differentiation and fiber regeneration. Necdin carries out this role mainly via interference with TNFα signaling at various levels, including regulation of expression of TNFR1 and p53, and regulation of the activity of caspase 3 and caspase 9. These data suggest that inhibition of muscle wasting using necdin is a feasible approach to treat cachexia in neoplastic patients.

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.

Silodosin and tadalafil have synergistic inhibitory effects on nerve-mediated contractions of human and rat isolated prostates.

Lower urinary tract symptoms (LUTS) in men with benign prostatic hyperplasia (BPH) are associated with erectile dysfunction. Alpha-1-adrenoceptor antagonists are effective drugs for treating symptomatic BPH. Clinical data show improvements in LUTS by phosphodiesterase 5 inhibitors. This study aimed to evaluate effects of silodosin, a highly selective α1A-adrenoceptor antagonist, alone or in combination with the phosphodiesterase 5 inhibitor tadalafil on contractions of isolated human and rat prostates. In organbath studies, effects of increasing concentrations of silodosin (1 nM-1 µM) and tadalafil (100 nM-100 µM) on contractions by electrical field stimulation or phenylephrine of human and rat prostate strip preparations were investigated. The combination silodosin and tadalafil reduced electrically-induced contractions of human prostate preparations better than single drugs alone. At any frequencies (1-32 Hz), inhibitory effects of combined therapy (P-values vs single drug) in human tissue were 26-42% (1 nM silodosin+100 nM tadalafil; P<0.05), 40-58% (10 nM silodosin+1 µM tadalafil; P<0.001-0.05), 56-67% (100 nM silodosin+10 µM tadalafil; P<0.01-0.05), and 33-55% (1 µM silodosin+100 µM tadalafil P<0.01-0.05). Similar findings were obtained in rat prostate preparations. In human and rat prostate tissue, the drug combination exerted similar inhibitory effect on phenylephrine contractions as silodosin alone. Silodosin plus tadalafil had greater potency than each drug alone to inhibit prostate contractions to electrical field stimulation but not to phenylephrine. This study supports the clinical application of a combination of an α1A-adrenoceptor antagonist and a phosphodiesterase 5 inhibitor for symptomatic BPH and suggests that the drug combination requires endogenous nerve-activity for optimal effect.

Effects by silodosin on the partially obstructed rat ureter in vivo and on human and rat isolated ureters

α1‐adrenoceptor (‐AR) antagonists may facilitate ureter stone passage in humans. We aimed to study effects by the α1A‐AR selective antagonist silodosin (compared to tamsulosin and prazosin) on ureter pressures in a rat model of ureter obstruction, and on contractions of human and rat isolated ureters.

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.

114 INHIBITION OF PERIPHERAL FATTY ACID AMIDE HYDROLASE AMELIORATES BLADDER OVERACTIVITY IN SPONTANEOUSLY HYPERTENSIVE RATS

treated rats had NVC. 2 out of 7 vehicleu-treated rats had incontinence episodes but sham or tadalafilu-treated rats did not. No differences in basal and threshold pressures, micturition intervals or volumes, or bladder capacity were recorded between groups. Micturition pressure amounted to 50 10, 45 18, and 25 5 cmH2O for sham, vehicle, and tadalafil (p 0.05). Tadalafil-treated rats had no residual volume (RV) whereas sham and vehicle-treated rats had RV of 0.002 0.0008 and 0.03 0.005ml. Compliance was 0.06 0.01, 0.04 0.01 (p 0.05), and 0.1 0.02 ml/cmH2O for sham, vehicle-, and tadalafiltreated rats. Nerve-mediated contractions amounted to 48 18 (sham), 29 6 (tadalafil) and 130 38 % of K (vehicle; p 0.05). Emax to carbachol amounted to 96 18 (sham), 125 25 (tadalafil), and 244 69 % of K (vehicle; p 0.05). Hypoxia was noted in the suburothelium with hydroxyprobe intenstities of 0.00016 0.00003 (sham), 0.00065 0.00003 (vehicle; p 0.05), and 0.00039 0.00006 (tadalafil). The actin/collagen 3 ratio was 0.4 0.08 (sham), 1.53 0.4 (vehicle; p 0.05), and 0.8 0.1 (tadalafil). Compared to vehicle, detrusor VAChT and CGRP nerves were reduced in CNI rats but were more frequent in tadalafil-treated rats compared to vehicle. CONCLUSIONS: CNI induced a BO with NVC without increased frequency. Detrusor from CNI rats had muscarinic supersensitivity, hypoxia and altered actin/collagen ratio. These changes were partially counteracted by 2-weeks daily treatment with tadalafil.

494 MESENCHYMAL STEM CELL MEDIATED CANCER THERAPY INHIBITS TUMOR GROWTH IN THE TRANSGENIC ADENOCARCINOMA OF THE MOUSE PROSTATE (TRAMP) MODEL

INTRODUCTION AND OBJECTIVES: Vectors that home tumors and micrometastases may be a unique tool to activate drugs selectively within a cancer site. Mesenchymal stem cells (MSC) as vectors for the prodrug-activating enzyme cytosine deaminase::uracil phosphoribosyltransferase (CD) convert the nontoxic 5-fluorocytosine (5FC, prodrug) into the antitumor drug 5-fluorouracil. Conversion occurs within the tumor mass thanks to the innate ability of MSC to target tumor lesions. We previously demonstrated that CD-MSC are effective in inhibiting growth of prostate tumor grafts in mice. Our objective was to further evaluate this therapeutic strategy in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model that better mimics onset and progression of human prostate disease. METHODS: After ethical approval, human adipose tissue (AT) MSC and mouse bone marrow (BM) MSC were used as vectors for CD. TRAMP mice were employed as a preclinical prostate cancer model. Autochthonous tumor volume was measured by magnetic resonance imaging (MRI) performed on a 7T preclinical scanner with multiplanar high resolution T2-weighted. Images post-processing was performed on MIPAV 12.5 software. Two millions CD-AT-MSC (first treatment) and CD-BM-MSC (second treatment) were injected into the tail vein. Prodrug (300 mg/kg/day) was administered intraperitoneally for 7-10 days after delivery of therapeutic cells. Mann-Whitney U test was used for comparisons RESULTS: TRAMP mice (ca. 26 weeks old) were divided into the following groups: CONTROL (received CD-MSC alone or 5FC alone) and TREATED (received CD-MSC plus 5FC). In the CONTROL group not all animals reached the time of the second MRI (some mice were sacrificed before second MRI for ethical reasons). Before therapy median TV was 91.19 mm 14.9 for CONTROL mice (n 6) and 106.25 mm 19.24 for TREATED mice (n 7). After therapy median TV was 106.77 mm 21.59 for CONTROL mice and 83.86 mm 14.09 for TREATED mice. We could therefore observe a median TV increase by 16.18% for the CONTROL mice and a median TV decrease by 28.72% for the TREATED mice (p 0.05). CONCLUSIONS: Systemic administration of CD-AT-MSC and CD-BM-MSC and subsequent prodrug treatment inhibit autochthonous prostate tumor growth. If confirmed in future larger studies, specifically engineered MSC cells may offer a new strategy to improve efficacy and selectivity of current antitumor therapies.

1575 NERVE-MEDIATED CONTRACTIONS OF HUMAN AND RAT ISOLATED PROSTATES ARE ADDITIVELY INHIBITED BY SILODOSIN AND TADALAFIL

malin (5%; 50 l per lobe) injection into bilateral ventral lobes of the prostate or E. coli (strain 1677) injection (0.2 mL of 1x 10 CFU/mL) into the prostatic urethra of male SD rats. After 5 days, ventral lobes of the prostate were harvested, and histopathology and real-time RT-PCR for pro-inflammatory cytokines were performed. Using laser-capture microdissection (LCM), glandular epithelial cells and stromal regions were separately isolated. Real-time RT-PCR was performed to examine mRNA levels of androgen-responsive (AR) genes in the epithelium and TGFcascade genes in the stroma. RESULTS: (1) Histopathology: In the formalin-injected prostate, inflammatory changes were observed focally. In the severe inflammation area, a large number of leukocytes were present in a slightly edematous stromal region and the epithelium shrank while other regions had almost normal appearance. In the E.coli-treated prostate, inflammation distributed diffusely throughout the prostate. Polymorphonuclear cells were filled in the ductal spaces and infiltrated into the highly edematous stroma, without morphological changes in the epithelium. (2) Cytokine levels: In the formalin-treated prostate, IL-1 , IL-6, and COX-2 mRNA were increased 3, 9, and 4-fold compared to control, respectively. In the E.coli-treated prostate, TNF, IL-1 , IL-6, and COX-2 mRNA increased 42, 149, 18 and 12-fold, respectively. (3) LCM: Epithelial cells from the area with no/little inflammation of formalin-treated prostate showed 2-3 fold increases in androgen receptors and AR genes such as Eaf2, ELL2, FKBP5, calreticulin and orinthine decarboxylase compared to control. In contrast, epithelial cells from E. coli-treated prostate or the severe inflammatory area of formalintreated prostate showed the reduction in these genes. In the stromal area from formalin or E. coli-treated prostate, TGFcascade genes such as TGF1, fibronectin and collagen-1 were up-regulated 2-5 fold. CONCLUSIONS: Prostatic inflammation changes expression of AR genes in the epithelium and TGFcascade genes in the stroma. Activation of TGFcascade genes in the inflammatory stroma as well as altered AR gene expression in the epithelium might be involved in the development of symptomatic BPH and/or prostate cancer.