Effects of benzo[a]pyrene on the reproductive axis: Impairment of kisspeptin signaling in human gonadotropin-releasing hormone primary neurons.

The neuroendocrine control of reproduction is strictly coordinated at the central level by the pulsatile release of gonadotropin-releasing hormone (GnRH) by the hypothalamic GnRH neurons. Alterations of the GnRH-network, especially during development, lead to long-term reproductive and systemic consequences, also causing infertility. Recent evidence shows that benzo[a]pyrene (BaP), a diffuse pollutant that can play a role as an endocrine disruptor, affects gonadal function and gamete maturation, whereas data demonstrating its impact at hypothalamic level are very scarce. This study investigated the effects of BaP (10 µM) in a primary cell culture isolated from the human fetal hypothalamus (hfHypo) and exhibiting a clear GnRH neuron phenotype. BaP significantly decreased gene and protein expression of both GnRH and kisspeptin receptor (KISS1R), the master regulator of GnRH neuron function. Moreover, BaP exposure increased phospho-ERK1/2 signaling, a well-known mechanism associated with KISS1R activation. Interestingly, BaP altered the electrophysiological membrane properties leading to a significant depolarizing effect and it also significantly increased GnRH release, with both effects being not affected by kisspeptin addition. In conclusion, our findings demonstrate that BaP may alter GnRH neuron phenotype and function, mainly interfering with KISS1R signaling and GnRH secretion and therefore with crucial mechanisms implicated in the central neuroendocrine control of reproduction.

Benzo[a]pyrene impairs the migratory pattern of human gonadotropin-releasing-hormone-secreting neuroblasts.

Benzo[a]pyrene (BaP) is a widespread pollutant that can act as an endocrine disrupting compound (EDC) and interferes with reproductive function. The central regulatory network of the reproductive system is mediated by gonadotropin-releasing hormone (GnRH) neurons, which originate in the olfactory placode and, during ontogenesis, migrate into the hypothalamus. Given the importance of the migratory process for GnRH neuron maturation, we investigated the effect of BaP (10 µM for 24 h) on GnRH neuroblasts isolated from the human fetal olfactory epithelium (FNCB4). BaP exposure significantly reduced the mRNA level of genes implicated in FNCB4 cell migration and affected their migratory ability. Our findings demonstrate that BaP may interfere with the central neuronal network controlling human reproduction affecting GnRH neuron maturation.

Tumor Necrosis Factor α Influences Phenotypic Plasticity and Promotes Epigenetic Changes in Human Basal Forebrain Cholinergic Neuroblasts.

TNFα is the main proinflammatory cytokine implicated in the pathogenesis of neurodegenerative disorders, but it also modulates physiological functions in both the developing and adult brain. In this study, we investigated a potential direct role of TNFα in determining phenotypic changes of a recently established cellular model of human basal forebrain cholinergic neuroblasts isolated from the nucleus basalis of Meynert (hfNBMs). Exposing hfNBMs to TNFα reduced the expression of immature markers, such as nestin and ß-tubulin III, and inhibited primary cilium formation. On the contrary, TNFα increased the expression of TNFα receptor TNFR2 and the mature neuron marker MAP2, also promoting neurite elongation. Moreover, TNFα affected nerve growth factor receptor expression. We also found that TNFα induced the expression of DNA-methylation enzymes and, accordingly, downregulated genes involved in neuronal development through epigenetic mechanisms, as demonstrated by methylome analysis. In summary, TNFα showed a dual role on hfNBMs phenotypic plasticity, exerting a negative influence on neurogenesis despite a positive effect on differentiation, through mechanisms that remain to be elucidated. Our results help to clarify the complexity of TNFα effects in human neurons and suggest that manipulation of TNFα signaling could provide a potential therapeutic approach against neurodegenerative disorders.

Oxadiazon affects the expression and activity of aldehyde dehydrogenase and acylphosphatase in human striatal precursor cells: A possible role in neurotoxicity.

Exposure to herbicides can induce long-term chronic adverse effects such as respiratory diseases, malignancies and neurodegenerative diseases. Oxadiazon, a pre-emergence or early post-emergence herbicide, despite its low acute toxicity, may induce liver cancer and may exert adverse effects on reproductive and on endocrine functions. Unlike other herbicides, there are no indications on neurotoxicity associated with long-term exposure to oxadiazon. Therefore, we have analyzed in primary neuronal precursor cells isolated from human striatal primordium the effects of non-cytotoxic doses of oxadiazon on neuronal cell differentiation and migration, and on the expression and activity of the mitochondrial aldehyde dehydrogenase 2 (ALDH2) and of the acylphosphatase (ACYP). ALDH2 activity protects neurons against neurotoxicity induced by toxic aldehydes during oxidative stress and plays a role in neurodegenerative conditions such as Alzheimer's disease and Parkinson's disease. ACYP is involved in ion transport, cell differentiation, programmed cell death and cancer, and increased levels of ACYP have been revealed in fibroblasts from patients affected by Alzheimer's disease. In this study we demonstrated that non-cytotoxic doses of oxadiazon were able to inhibit neuronal striatal cell migration and FGF2- and BDNF-dependent differentiation towards neuronal phenotype, and to inhibit the expression and activity of ALDH2 and to increase the expression and activity of ACYP2. In addition, we have provided evidence that in human primary neuronal precursor striatal cells the inhibitory effects of oxadiazon on cell migration and differentiation towards neuronal phenotype were achieved through modulation of ACYP2. Taken together, our findings reveal for the first time that oxadiazon could exert neurotoxic effects by impairing differentiative capabilities of primary neuronal cells and indicate that ALDH2 and ACYP2 are relevant molecular targets for the neurotoxic effects of oxadiazon, suggesting a potential role of this herbicide in the onset of neurodegenerative diseases.

Human fetal adrenal cells retain age-related stem- and endocrine-differentiation potential in culture.

The adrenal gland is a multiendocrine organ with a steroidogenic mesenchymal cortex and an inner catecholamine-producing medulla of neuroendocrine origin. After embryonic development, this plastic organ undergoes a functional postnatal remodeling. Elucidating these complex processes is pivotal for understanding the early bases of functional endocrine disorders and tumors affecting the mature gland. We developed an in vitro human adrenal cell model derived from fetal adrenal specimens at different gestational ages, consisting of neuroendocrine and cortical components and expressing the zona and functional markers of the original fetal organ. These cortical and neuroendocrine progenitor cells retain in vitro an intrinsic gestational-age-related differentiation and functional program. In vitro these cells spontaneously form 3-dimensional structure organoids with a structure similar to the fetal gland. The organoids show morphofunctional features and adrenal steroidogenic factor, steroid acute regulatory, cytochrome-P450-17A1, dosage-sensitive, sex-reversal, adrenal hypoplasia-critical region on chromosome X protein , NOTCH1, and nephroblastoma overexpressed/cysteine-rich protein 61/connective tissue growth factor/nephroblastoma overexpressed gene-3; stem (BMI1, nestin); and chromaffin (chromogranin A, tyrosine hydroxylase) markers similar to those of the populations of origin. This in vitro human adrenal system represents a unique but preliminar model for investigating the pathophysiological processes underlying physiologic adrenal remodeling and pathologic alterations involved in organ hypo- and hyperplasia and cancer.-Poli, G., Sarchielli, E., Guasti, D., Benvenuti, S., Ballerini, L., Mazzanti, B., Armignacco, R., Cantini, G., Lulli, M., Chortis, V., Arlt, W., Romagnoli, P., Vannelli, G. B., Mannelli, M., Luconi, M. Human fetal adrenal cells retain age-related stem- and endocrine-differentiation potential in culture.

INT-767 prevents NASH and promotes visceral fat brown adipogenesis and mitochondrial function.

The bile acid receptors, farnesoid X receptor (FXR) and Takeda G-protein-coupled receptor 5 (TGR5), regulate multiple pathways, including glucose and lipid metabolism. In a rabbit model of high-fat diet (HFD)-induced metabolic syndrome, long-term treatment with the dual FXR/TGR5 agonist INT-767 reduces visceral adipose tissue accumulation, hypercholesterolemia and nonalcoholic steatohepatitis. INT-767 significantly improves the hallmarks of insulin resistance in visceral adipose tissue (VAT) and induces mitochondrial and brown fat-specific markers. VAT preadipocytes isolated from INT-767-treated rabbits, compared to preadipocytes from HFD, show increased mRNA expression of brown adipogenesis markers. In addition, INT-767 induces improved mitochondrial ultrastructure and dynamic, reduced superoxide production and improved insulin signaling and lipid handling in preadipocytes. Both in vivo and in vitro treatments with INT-767 counteract, in preadipocytes, the HFD-induced alterations by upregulating genes related to mitochondrial biogenesis and function. In preadipocytes, INT-767 behaves mainly as a TGR5 agonist, directly activating dose dependently the cAMP/PKA pathway. However, in vitro experiments also suggest that FXR activation by INT-767 contributes to the insulin signaling improvement. INT-767 treatment counteracts HFD-induced liver histological alterations and normalizes the increased pro-inflammatory genes. INT-767 also induces a significant reduction of fatty acid synthesis and fibrosis markers, while increasing lipid handling, insulin signaling and mitochondrial markers. In conclusion, INT-767 significantly counteracts HFD-induced liver and fat alterations, restoring insulin sensitivity and prompting preadipocytes differentiation toward a metabolically healthy phenotype.

Cortical and spinal conditioned media modify the inward ion currents and excitability and promote differentiation of human striatal primordium.

Human striatal precursor cells (HSPs) isolated from ganglionic eminence may differentiate in electrophysiologically functional excitable neuron-like cells and a number of endogenous molecules such as hormones, neurotransmitters or growth factors can actually regulate neuronal growing and differentiation. The purpose of this research was to assess, by electrophysiological and immunocytochemical analysis, if the type of culture medium could specifically impact on the neuronal differentiation potential of HSPs. Accordingly, HSPs were maintained in different inductive media such as cortical and spinal cord conditioned media, and we estimated the possible changes in the main ion currents, excitability and expression of neuronal markers indicative of neuronal differentiation. Our results have shown that 36 h exposure to each of the conditioned media, with their blend of autocrine and paracrine growth factors, was able to modify significantly the electrophysiological membrane properties and the functional expression of inward ionic currents in selected neuronal HSPs. Moreover, although both types of conditioned media determined neuronal maturation (increased neuritogenesis and increased expression of neuronal and striatal markers), each of them leads to the occurrence of different functional features. Particularly, the spinal medium caused a stronger depolarization of the membrane potential and significantly increased the amplitude of Na+ current as well as L- and N- type Ca2+ currents, definitely modifying their kinetics. In contrast, the cortical medium mainly caused a significant and more marked increase of the membrane conductance and time constant values. These results strongly support the plasticity of our cellular model that, although already committed towards a specific phenotype, it can be differently affected by the conditioned media, thereby resulting functionally modifiable according to environmental cues.

Anti-fibrotic effects of chronic treatment with the selective FXR agonist obeticholic acid in the bleomycin-induced rat model of pulmonary fibrosis.

Farnesoid X receptor (FXR) activation by obeticholic acid (OCA) has been demonstrated to inhibit inflammation and fibrosis development in liver, kidney and intestine in multiple disease models. FXR activation has also been demonstrated to suppress the inflammatory response and to promote lung repair after lung injury. This study investigated the protective effects of OCA treatment (3 or 10mg/kg/day) on inflammation, tissue remodeling and fibrosis in the bleomycin-induced pulmonary fibrosis rat model. Effects of OCA treatment on morphological and molecular alterations of the lung, as well as remodeling of the alveoli and the right ventricle were also evaluated. Lung function was assessed by measuring airway resistance to inflation. In the acute phase (7days), bleomycin promoted an initial thickening and fibrosis of the lung interstitium, with upregulation of genes related to epithelial proliferation, tissue remodeling and hypoxia. At 28days, an evident increase in the deposition of collagen in the lungs was observed. This excessive deposition was accompanied by an upregulation of transcripts related to the extracellular matrix (TGFß1, SNAI1 and SNAI2), indicating lung fibrosis. Administration of OCA protected against bleomycin-induced lung damage by suppressing molecular mechanisms related to epithelial-to-mesenchymal transition (EMT), inflammation and collagen deposition, with a dose-dependent reduction of proinflammatory cytokines such as IL-1ß and IL-6, as well as TGF-ß1 and SNAI1 expression. Pirfenidone, a recently approved treatment for idiopathic pulmonary fibrosis (IPF), significantly counteracted bleomycin-induced pro-fibrotic genes expression, but did not exert significant effects on IL-1ß and IL-6. OCA treatment in bleomycin-challenged rats also improved pulmonary function, by effectively normalizing airway resistance to inflation and lung stiffness in vivo. Results with OCA were similar, or even superior, to those obtained with pirfenidone. In conclusion, our results suggest an important protective effect of OCA against bleomycin-induced lung fibrosis by blunting critical mediators in the pathogenesis of IPF.

Potential role for the VDR agonist elocalcitol in metabolic control: Evidences in human skeletal muscle cells.

Vitamin D plays a pivotal role to maintain skeletal muscle integrity and health. Vitamin D deficiency characterizes inflammatory myopathy (IM) and diabetes, often overlapping diseases involving skeletal muscle damage. Vitamin D receptor (VDR) agonists likely exert beneficial effects in both IM and metabolic disturbances. We aim to evaluate in vitro the effect of elocalcitol, a non-hypercalcemic VDR agonist, on the biomolecular metabolic machinery of human skeletal muscle cells (Hfsmc), vs. insulin (I). We analyzed GLUT4, Flotillin-1, Caveolin-3 and Caveolin-1 cell expression/localization; mTOR, AKT, ERK and 4E-BP1 phosphorylation; IL-6 myokine release; VDR expression. We investigated in vivo vitamin D status in IM subjects, evaluating VDR muscular expression and serum vitamin D with metabolism-related parameters, as glycemia, triglycerides, cholesterol, resistin and adiponectin. In Hfsmc, elocalcitol exerted an I-like effect, promoting GLUT4 re-localization in Flotillin-1, Caveolin-3 and Caveolin-1 positive sites and mTOR, AKT, ERK, 4E-BP1 activation; it enhanced IL-6 myokine release. IM subjects, all normoglycemic, showed VDR/vitamin D deficiency that, together with high lipidemic and resistin profile, possibly increases the risk to develop metabolic diseases. VDR agonists as elocalcitol may be therapeutic tools for skeletal muscle integrity/function maintenance, an indispensable condition for health homeostasis.

Cardiopulmonary protective effects of the selective FXR agonist obeticholic acid in the rat model of monocrotaline-induced pulmonary hypertension.

Farnesoid X receptor (FXR) activation by obeticholic acid (OCA) has been demonstrated to inhibit inflammation and fibrosis development and even induce fibrosis regression in liver, kidney and intestine in multiple disease models. OCA also inhibits liver fibrosis in nonalcoholic steatohepatitis patients. FXR activation has also been demonstrated to suppress the inflammatory response and to promote lung repair after lung injury. This study investigated the effects of OCA treatment (3, 10 or 30mg/kg, daily for 5days a week, for 7 and/or 28 days) on inflammation, tissue remodeling and fibrosis in the monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) rat model. Treatment with OCA attenuated MCT-induced increased pulmonary arterial wall thickness and right ventricular hypertrophy, by i) blunting pathogenic inflammatory mechanisms (downregulation of interleukin 6, IL-6, and monocyte chemoattractant protein-1, MCP-1) and ii) enhancing protective mechanisms counteracting fibrosis and endothelial/mesenchymal transition. MCT-injected rats also showed a marked decrease of pulmonary artery responsiveness to both endothelium-dependent and independent relaxant stimuli, such as acetylcholine and a nitric oxide donor, sodium nitroprusside. Administration of OCA (30mg/kg) normalized this decreased responsiveness. Accordingly, OCA treatment induced profound beneficial effects on lung histology. In particular, both OCA doses markedly reduced the MCT-induced medial wall thickness increase in small pulmonary arteries. To evaluate the objective functional improvement by OCA treatment of MCT-induced PAH, we performed a treadmill test and measured duration of exercise. MCT significantly reduced, and OCA normalized treadmill endurance. Results with OCA were similar, or even superior, to those obtained with tadalafil, a well-established treatment of PAH. In conclusion, OCA treatment demonstrates cardiopulmonary protective effects, modulating lung vascular remodeling, reducing right ventricular hypertrophy and significantly improving exercise capacity. Thus, OCA can restore the balance between relaxant and contractile pathways in the lung, promoting cardiopulmonary protective actions.

Searching for Classical Brown Fat in Humans: Development of a Novel Human Fetal Brown Stem Cell Model.

The potential therapeutic applications of targeting brown adipose tissue open new clinical avenues in fighting against metabolic pathologies. However, due to the limited extension in adult humans of brown depots, which are dramatically reduced after birth, solid cell models to study human brown adipogenesis and its regulatory factors in pathophysiology are urgently needed. Here, we generated a novel human model of brown adipose stem cells, hfB-ASC, derived for the first time from fetal interscapular brown fat depots. Besides the characterization of their stem and classical brown adipose properties, we demonstrated that these cells retain a specific intrinsic differentiation program to functional brown adipocytes, even spontaneously generating organoid structures with brown features. Moreover, for the first time, we investigated the thermogenic and electrophysiological activity of the in vitro-derived fetal brown adipocytes compared to their undifferentiated precursors hfB-ASC, in basal and norepinephrine-induced conditions. In conclusion, from interscapular brown fat of the human fetus we developed and functionally characterized a novel physiological brown adipose stem cell model early programmed to brown differentiation, which may represent a unique opportunity for further studies on brown adipogenesis processes in humans as well as the most suitable target to study novel therapeutic approaches for stimulating brown activity in metabolic pathologies. Stem Cells 2016;34:1679-1691.

Tadalafil reduces visceral adipose tissue accumulation by promoting preadipocytes differentiation towards a metabolically healthy phenotype: Studies in rabbits.

Development of metabolically healthy adipocytes within dysfunctional adipose tissue may represent an attractive way to counteract metabolic syndrome (MetS). In an experimental animal model of high fat diet (HFD)-induced MetS, in vivo, long- and short-term tadalafil treatments were able to reduce visceral adipose tissue (VAT) accumulation and hypertriglyceridemia, and to induce the expression in VAT of the brown fat-specific marker, uncoupling protein 1 (UCP1). VAT preadipocytes (PAD), isolated from the tadalafil-treated HFD rabbits, showed: i) a multilocular morphology; ii) an increased expression of brown fat-specific genes (such as UCP1 and CIDEA); iii) improved mitochondrial structure and dynamic and reduced superoxide production; iv) improved insulin sensitivity. Similar effects were obtained after in vitro tadalafil treatment in HFD rPAD. In conclusion, tadalafil counteracted HFD-associated VAT alterations, by restoring insulin-sensitivity and prompting preadipocytes differentiation towards a metabolically healthy phenotype.

Differential expression of vascular endothelial growth factor in human fetal skeletal site-specific tissues: Mandible versus femur.

Vascular endothelial growth factor (VEGF) is a well-known mediator that signals through pathways in angiogenesis and osteogenesis. Angiogenesis and bone formation are coupled during either skeletal development or bone remodeling and repair occurring in postnatal life. In this study, we examined for the first time the expression of VEGF in human fetal mandibular and femoral bone in comparison with the respective adult tissues. Similarly to other craniofacial bones, but at variance with the axial and appendicular skeleton, during development mandible does not arise from mesoderm but neural crest cells of the neuroectoderm germ layer, and undergoes intramembranous instead of endochondral ossification. By quantitative real-time PCR technique, we could show that VEGF gene expression levels were significantly higher in fetal than in adult samples, especially in femoral tissue. Western blotting analysis confirmed higher protein expression of VEGF in the fetal femur respect to the mandible. Moreover, immunohistochemistry revealed that in both fetal tissues VEGF expression was mainly localized in pre- and osteoblasts. Differential expression of VEGF in femoral and mandibular bone tissues could be related to their different structure, function and development during organogenesis.

Early effects of corneal collagen cross-linking by iontophoresis in ex vivo human corneas.

PURPOSE: The purpose was to investigate the early modifications induced by collagen cross-linking by iontophoresis of riboflavin (ionto-CXL) in ex vivo human corneas by evaluating different protocols of UVA irradiation. METHODS: In this experimental study 46 ex vivo human corneas obtained from the Eye Bank of Mestre (Italy) were divided in different groups: six were utilized as control (CTL); eight were treated with ionto-CXL at 3 mW/cm(2) power for 30 min (I-3); eight were treated with ionto-CXL at 10 mW/cm(2) for 9 min (I-10); eight were treated with iontophoretic delivery of riboflavin only (I-0); eight were treated with the standard CXL at 3 mW/cm(2) for 30 min (S-3); and eight were treated with CXL at 10 mW/cm(2) for 9 min (S-10). All samples were evaluated by haematoxylin-eosin staining and immunohistochemical analysis using different markers (Connexin 43, CD34, Collagen I, TUNEL assay). Western blot analysis, utilizing Bax and Ki67 primary antibodies, for detection of keratocyte apoptosis and proliferation, respectively, was also performed. RESULTS: No endothelial damage was evidenced in the treated groups. In I-10 corneas the epithelial layers were not always well-preserved. Anterior stroma showed an uneven distribution and numerical reduction of keratocytes as well as increased apoptosis; a reduced subepithelial interweaving of collagen I fibers was observed. In S-3 and S-10 the changes induced by treatments were similar to I-10. I-3 and I-0 showed no significant changes with respect to the control group. CONCLUSIONS: In the ionto-CXL at 10 mW/cm(2) group occurred the main morphological and biomolecular changes. This experimental study suggests that iontophoresis can be considered a non-invasive potential delivery tool for riboflavin penetration in corneal stroma during CXL.

Expression of sialic acids in human adult skeletal muscle tissue.

Investigations mostly in animal models have shown a role of sialic acid in the morphology and functionality of skeletal muscle during development and adult life. Several studies in humans have been performed regarding changes in sialic acid expression in a particular pathology, hereditary inclusion body myopathy, leading to muscular weakness and atrophy, with a similar phenomenon appearing also in sarcopenia of aging. In this study the expression of monomeric and polymeric sialic acids was evaluated in human skeletal muscle during adult life. Surgical biopsies of the Quadriceps femoris muscle from men aged 18-25 years (young group; n=8) and men aged 72-78 (elderly group; n=10) were collected for analysis. Expression of sialic acids was evaluated using lectin histochemistry, associated with enzymatic and chemical treatments to characterize monomeric and polymeric sialic acids. The polysialic acid expression was also evaluated by immunohistochemistry. Various types of sialic acid in the muscle tissue, in different amounts in the study groups, were detected. Monomeric sialic acids decreased in the elderly group compared with the young group, whereas polysialic acid increased. Sialic acid acetylation was present only in the young group. These findings demonstrated that changes in the expression of sialic acids in skeletal muscle tissue may be related to morphofunctional modifications occurring during aging.

Tadalafil effect on metabolic syndrome-associated bladder alterations: an experimental study in a rabbit model.

INTRODUCTION: Metabolic syndrome (MetS) and lower urinary tract symptoms (LUTS) are often associated. Bladder detrusor hyper-contractility-a major LUTS determinant-is characterized by increased Ras homolog gene family, member A/Rho-associated protein kinase (RhoA/ROCK) signaling, which is often upregulated in MetS. AIM: This study investigated the effects of tadalafil dosing on RhoA/ROCK signaling in bladder, in a rabbit model of high-fat diet (HFD)-induced MetS. METHODS: Adult male rabbits feeding a HFD for 12 weeks. A subset of HFD animals was treated with tadalafil (2 mg/kg/day, 1 week: the last of the 12 weeks) and compared with HFD and control (feeding a regular diet) rabbits. MAIN OUTCOME MEASURES: In vitro contractility studies to evaluate the relaxant effect of the selective ROCK inhibitor, Y-27632, in carbachol precontracted bladder strips. Evaluation of RhoA activation by its membrane translocation. Immunohistochemistry for ROCK expression has been performed to evaluate ROCK expression in bladder from the different experimental groups. mRNA expression of inflammation, pro-fibrotic markers by quantitative RT-PCR has been performed to evaluate the effect of tadalafil on MetS-induced inflammation and fibrosis within the bladder. The in vitro effect of tadalafil on RhoA/ROCK signaling in bladder smooth muscle cells was evaluated by using chemotaxis assay. RESULTS: Bladder strips from HFD rabbits showed hyper-responsiveness to Y-27632, indicating RhoA/ROCK overactivity in HFD bladder compared with matched controls. Accordingly, the fraction of activated (translocated to the membrane) RhoA as well as ROCK expression are increased in HFD bladder. Tadalafil dosing normalized HFD-induced bladder hypersensitivity to Y-27632, by reducing RhoA membrane translocation and ROCK overexpression. Tadalafil dosing reduced mRNA expression of inflammatory, pro-fibrotic, and hypoxia markers. A direct inhibitory effect of tadalafil on RhoA/ROCK signaling in bladder smooth muscle cell was demonstrated by using chemotaxis assay. Pre-treatment with tadalafil inhibited both basal and PDGF-induced migration of bladder smooth muscle cells. CONCLUSIONS: Tadalafil dosing reduced RhoA/ROCK signaling and smooth muscle overactivity in an animal model of MetS-associated bladder alterations. Our findings suggest a novel mechanism of action of tadalafil in alleviating LUTS in MetS patients.

Nonalcoholic steatohepatitis as a novel player in metabolic syndrome-induced erectile dysfunction: an experimental study in the rabbit.

A pathogenic link between erectile dysfunction (ED) and metabolic syndrome (MetS) is now well established. Nonalcoholic steatohepatitis (NASH), the hepatic hallmark of MetS, is regarded as an active player in the pathogenesis of MetS-associated cardiovascular disease (CVD). This study was aimed at evaluating the relationship between MetS-induced NASH and penile dysfunction. We used a non-genomic, high fat diet (HFD)-induced, rabbit model of MetS, and treated HFD rabbits with testosterone (T), with the selective farnesoid X receptor (FXR) agonist obeticholic acid (OCA), or with the anti-TNFα mAb infliximab. Rabbits fed a regular diet were used as controls. Liver histomorphological and gene expression analysis demonstrated NASH in HFD rabbits. Several genes related to inflammation (including TNFα), activation of stellate cells, fibrosis, and lipid metabolism parameters were negatively associated to maximal acetylcholine (Ach)-induced relaxation in penis. When all these putative liver determinants of penile Ach responsiveness were tested as covariates in a multivariate model, only the association between hepatic TNFα expression and Ach response was confirmed. Accordingly, circulating levels of TNFα were increased 15-fold in HFD rabbits. T and OCA dosing in HFD rabbits both reduced TNFα liver expression and plasma levels, with a parallel increase of penile eNOS expression and responsiveness to Ach. Also neutralization of TNFα with infliximab treatment fully normalized HFD-induced hypo-responsiveness to Ach, as well as responsiveness to vardenafil, a phosphodiesterase type 5 inhibitor. Thus, MetS-induced NASH in HFD rabbits plays an active role in the pathogenesis of ED, likely through TNFα, as indicated by treatments reducing liver and circulating TNFα levels (T or OCA), or neutralizing TNFα action (infliximab), which significantly improve penile responsiveness to Ach in HFD rabbits.

Large-sized Fetal Striatal Grafts in Huntington's Disease Do Stop Growing: Long-term Monitoring in the Florence Experience.

Development of six large nodules of solid tissue after bilateral human fetal striatal transplantation in four Huntington's disease patients has raised concern about the safety of this experimental therapy in our setting. We investigated by serial MRI-based volumetric analysis the growth behaviour of such grafts. After 33-73 months from transplantation the size of five grafts was stable and one graft showed a mild decrease in size. Signs neither of intracranial hypertension nor of adjuctive focal neurological deficit have ever been observed. This supports long-term safety of the grafting procedure at our Institution.

Fetal striatal grafting slows motor and cognitive decline of Huntington's disease.

OBJECTIVE: To assess the clinical effect of caudate-putaminal transplantation of fetal striatal tissue in Huntington's disease (HD). METHODS: We carried out a follow-up study on 10 HD transplanted patients and 16 HD not-transplanted patients. All patients were evaluated with the Unified HD Rating Scale (UHDRS) whose change in motor, cognitive, behavioural and functional capacity total scores were considered as outcome measures. Grafted patients also received morphological and molecular neuroimaging. RESULTS: Patients were followed-up from disease onset for a total of 309.3 person-years (minimum 5.3, median 11.2 years, maximum 21.6 years). UHDRS scores have been available since 2004 (median time of 5.7 years since onset, minimum zero, maximum 17.2 years). Median post-transplantation follow-up was 4.3 years, minimum 2.8, maximum 5.1 years. Adjusted post-transplantation motor score deterioration rate was reduced compared to the pretransplantation period, and to that of not-transplanted patients by 0.9 unit/years (95% CI 0.2 to 1.6). Cognitive score deterioration was reduced of 2.7 unit/years (95% CI 0.1 to 5.3). For grafted patients the 2-year post-transplantation [(18)F]fluorodeoxyglucose positron emission tomography (PET) showed striatal/cortical metabolic increase compared to the presurgical evaluation; 4-year post-transplantation PET values were slightly decreased, but remained higher than preoperatively. [(123)I]iodobenzamide single photon emission CT demonstrated an increase in striatal D2-receptor density during postgrafting follow-up. CONCLUSIONS: Grafted patients experienced a milder clinical course with less pronounced motor/cognitive decline and associated brain metabolism improvement. Life-time follow-up may ultimately clarify whether transplantation permanently modifies the natural course of the disease, allowing longer sojourn time at less severe clinical stage, and improvement of overall survival.

The vitamin D receptor agonist BXL-01-0029 as a potential new pharmacological tool for the treatment of inflammatory myopathies.

OBJECTIVE: This study aims to investigate in vitro the effect of the VDR agonist BXL-01-0029 onto IFNγ/TNFα-induced CXCL10 secretion by human skeletal muscle cells compared to elocalcitol (VDR agonist), methylprednisolone, methotrexate, cyclosporin A, infliximab and leflunomide; to assess in vivo circulating CXCL10 level in subjects at time of diagnosis with IMs, before therapy, together with TNFα, IFNγ, IL-8, IL-6, MCP-1, MIP-1ß and IL-10, vs. healthy subjects. METHODS: Human fetal skeletal muscle cells were used for in vitro studies; ELISA and Bio-Plex were used to measure cell supernatant and IC50 determination or serum cytokines; Western blot and Bio-Plex were for cell signaling analysis. RESULTS: BXL-01-0029 decreased with the highest potency IFNγ/TNFα-induced CXCL10 protein secretion and targeted cell signaling downstream of TNFα in human skeletal muscle cells; CXCL10 level was the highest in sera of subjects diagnosed with IMs before therapy and the only one significantly different vs. healthy controls. CONCLUSIONS: Our in vitro and in vivo data, while confirm the relevance of CXCL10 in IMs, suggested BXL-01-0029 as a novel pharmacological tool for IM treatment, hypothetically to be used in combination with the current immunosuppressants to minimize side effects.