Testosterone positively regulates vagina NO-induced relaxation: an experimental study in rats.

PURPOSE: Female sexual response involves a complex interplay between neurophysiological mechanisms and the nitric oxide (NO)-mediated relaxation of clitoris and vagina. The aim of this study was to evaluate sex steroids regulation of the relaxant pathway in vagina, using a validated animal model. METHODS: Subgroups of OVX Sprague-Dawley rats were treated with 17ß-estradiol, testosterone, or testosterone and letrozole, and compared with a group of intact animals. Masson's trichrome staining was performed for morphological evaluation of the distal vaginal wall, in vitro contractility studies investigated the effect of OVX and in vivo treatments on vaginal smooth muscle activity. RNA from vaginal tissue was analyzed by semi-quantitative RT-PCR. RESULTS: Immunohistochemical analysis showed that OVX induced epithelial and smooth muscle structural atrophy, testosterone and testo + letrozole increased the muscle bundles content and organization without affecting the epithelium while 17ß-estradiol mediated the opposite effects. In vitro contractility studies were performed on noradrenaline pre-contracted vaginal strips from each experimental group. Acetylcholine (0.001-10 µM) stimulation induced a concentration-dependent relaxation, significantly reduced by NO-synthase inhibitor L-NAME and by guanylate cyclase inhibitor ODQ. OVX resulted in a decreased responsiveness to acetylcholine, restored by testosterone, with or without letrozole, but not by 17ß-estradiol. OVX sensitivity to the NO-donor SNP was higher than in the control. Vardenafil, a PDE5 inhibitor, enhanced SNP effect in OVX + testosterone as well as in control, as supported by RNA expression analysis. CONCLUSIONS: Our study demonstrates that testosterone improves the NO-mediated smooth muscle vaginal cells relaxation confirming its role in maintaining the integrity of muscular relaxant machinery.

Treatment potential of LPCN 1144 on liver health and metabolic regulation in a non-genomic, high fat diet induced NASH rabbit model.

PURPOSE: Low free testosterone (T) level in men is independently associated with presence and severity of Non-Alcoholic Steatohepatitis (NASH). The histological and molecular effects of oral testosterone prodrug LPCN 1144 treatment on hepatic fibrosis and NASH features are unknown. A metabolic syndrome-induced NASH model in rabbits consuming high fat diet (HFD) has been previously used to assess treatment effects of injectable T on hepatic fibrosis and NASH features. Here we present results on LPCN 1144 in this HFD-induced, NASH preclinical model. METHODS: Male rabbits were randomly assigned to five groups: regular diet (RD), HFD, HFD + 1144 vehicle (HFD + Veh), HFD + 1144 (1144), and HFD + 1144 + α-tocopherol (1144 + ALPHA). Rabbits were sacrificed after 12 weeks for liver histological, biochemical and genetic analyses. Histological scores were obtained through Giemsa (inflammation), Masson's trichrome (steatosis and ballooning), and Picrosirius Red (fibrosis) staining. RESULTS: Compared to RD, HFD and HFD + Veh significantly worsened NASH features and hepatic fibrosis. Considering HFD and HFD + Veh arms, histological and biomarker features were not significantly different. Both 1144 and 1144 + ALPHA arms improved mean histological scores of NASH as compared to HFD arm. Importantly, percentage of fibrosis was improved in both 1144 (p < 0.05) and 1144 + ALPHA (p = 0.05) treatment arms vs. HFD. Both treatment arms also reduced HFD-induced inflammation and fibrosis mRNA markers. Furthermore, 1144 treatments significantly improved HFD-induced metabolic dysfunctions. CONCLUSIONS: Histological and biomarker analyses demonstrate that LPCN 1144 improved HFD-induced hepatic fibrosis and NASH biochemical, biomolecular and histochemical features. These preclinical findings support a therapeutic potential of LPCN 1144 in the treatment of NASH and of hepatic fibrosis.

A unique neuroendocrine cell model derived from the human foetal neural crest.

PURPOSE: Nowadays, no human neuroendocrine cell models derived from the neural crest are available. In this study, we present non-transformed long-term primary Neural Crest Cells (NCCs) isolated from the trunk region of the neural crest at VIII-XII gestational weeks of human foetuses obtained from voluntary legal abortion. METHODS AND RESULTS: In NCC, quantitative real-time RT PCR demonstrated the expression of neural crest specifier genes, such as Snail1, Snail2/SLUG, Sox10, FoxD3, c-Myc, and p75NTR. Moreover, these cell populations expressed stemness markers (such as Nanog and nestin), as well as markers of motility and invasion (TAGLN, MMP9, CXCR4, and CXCR7), and of neuronal/glial differentiation (MAP2, GFAP, SYP, and TAU). Functional analysis demonstrated that these cells not only possessed high migration properties, but most importantly, they expressed markers of sympatho-adrenal lineage, such as ASCL1 and tyrosine hydroxylase (TH). Moreover, the expression of TH increased after the induction with two different protocols of differentiation towards neuronal and sympatho-adrenal phenotypes. Finally, exposure to conditioned culture media from NCC induced a mature phenotype in a neuronal cell model (namely SH-SY5Y), suggesting that NCC may also act like Schwann precursors. CONCLUSION: This unique human cell model provides a solid tool for future studies addressing the bases of human neural crest-derived neuroendocrine tumours.

Therapeutic effects of the selective farnesoid X receptor agonist obeticholic acid in a monocrotaline-induced pulmonary hypertension rat model.

BACKGROUND: Activation of the farnesoid X receptor (FXR), a member of the nuclear receptor steroid superfamily, leads to anti-inflammatory and anti-fibrotic effects in several tissues, including the lung. We have recently demonstrated a protective effect of the farnesoid X receptor (FXR) agonist obeticholic acid (OCA) in rat models of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) and bleomycin-induced pulmonary fibrosis. The aim of the present study was to investigate whether the positive effects of OCA treatment could be exerted also in established MCT-induced PAH, i.e., starting treatment 2 weeks after MCT administration. METHODS: Rats with MCT-induced PAH were treated, 2 weeks after MCT administration, with OCA or tadalafil for two additional weeks. Pulmonary functional tests were performed at week 2 (before treatment) and four (end of treatment). At the same time points, lung morphological features and expression profile of genes related to smooth muscle relaxation/contraction and tissue remodeling were also assessed. RESULTS: 2 weeks after MCT-induced injury, the treadmill resistance (a functional parameter related to pulmonary hypertension) was significantly decreased. At the same time point, we observed right ventricular hypertrophy and vascular remodeling, with upregulation of genes related to inflammation. At week 4, we observed a further worsening of the functional and morphological parameters, accompanied by dysregulation of inflammatory and extracellular matrix markers mRNA expression. Administration of OCA (3 or 10 mg/kg/day), starting 2 weeks after MCT-induced injury, significantly improved pulmonary function, effectively normalizing the exercise capacity. OCA also reverted most of the lung alterations, with a significant reduction of lung vascular wall thickness, right ventricular hypertrophy, and restoration of the local balance between relaxant and contractile pathways. Markers of remodeling pathways were also normalized by OCA treatment. Notably, results with OCA treatment were similar, or even superior, to those obtained with tadalafil, a recently approved treatment for pulmonary hypertension. CONCLUSIONS: The results of this study demonstrate a significant therapeutic effect of OCA in established MCT-induced PAH, improving exercise capacity associated with reduction of right ventricular hypertrophy and lung vascular remodeling. Thus, OCA dosing in a therapeutic protocol restores the balance between relaxant and contractile pathways in the lung, promoting cardiopulmonary protective actions in MCT-induced PAH.

Therapeutic effects of obeticholic acid (OCA) treatment in a bleomycin-induced pulmonary fibrosis rat model.

PURPOSE: We recently demonstrated a protective effect of the farnesoid X receptor agonist obeticholic acid (OCA) in rat models of bleomycin-induced pulmonary fibrosis (PF). Aim of the present study was to investigate whether the positive effects of OCA treatment are apparent also on ongoing bleomycin-induced PF, i.e., after 2 weeks of bleomycin administration. METHODS: Bleomycin-induced PF rats were treated 2 weeks after bleomycin administration with OCA or pirfenidone for two additional weeks. Pulmonary function test was performed at 2 and 4 weeks in all experimental groups. At the same time points, lung morphological features and mRNA expression profile of genes related to fibrosis, inflammation and epithelial-mesenchymal transition were also assessed. RESULTS: After 2 weeks, bleomycin significantly increased the pressure at the airway opening (PAO), a functional parameter related to fibrosis-induced lung stiffness, and induced diffuse lung interstitium fibrosis, with upregulation of inflammation (IL1ß, MCP1) and tissue remodeling (COL1A1, COL3A1, ET1, MMP7, PDGFa, αSMA, SNAI1) markers. At week four, a further increase of lung fibrosis and PAO was observed, accompanied by upregulation of extracellular matrix-related mRNA expression. OCA administration, even after the establishment of PF, significantly improved pulmonary function, normalizing PAO, and reverted the bleomycin-induced lung alterations, with significant reduction of markers of inflammation (CD206, COX2, HIF1, IL1ß, MCP1), epithelial proliferation (CTGF, PDGFa) and fibrosis (COL1A1, COL3A1, ET1, FN1, MMPs, αSMA, SNAIs, TGFß1, TIMPs). Results with OCA were similar or superior to those obtained with pirfenidone. CONCLUSIONS: In conclusion, our results demonstrate a significant therapeutic effect of OCA in already established PF.

Opposite effects of tamoxifen on metabolic syndrome-induced bladder and prostate alterations: a role for GPR30/GPER?

BACKGROUND: BPH and LUTS have been associated to obesity, hypogonadism, and metabolic syndrome (MetS). MetS-induced prostate and bladder alterations, including inflammation and tissue remodeling, have been related to a low-testosterone and high-estrogen milieu. In addition to ERs, GPR30/GPER is able to mediate several estrogenic non-genomic actions. METHODS: Supplementing a subgroup of MetS rabbits with tamoxifen, we analyzed the in vivo effects on MetS-induced prostate and bladder alterations. The effects of selective ER/GPER ligands and GPER silencing on prostate inflammation were also studied in vitro using hBPH cells. RESULTS: ERα, ERß, and PR expression was upregulated in MetS bladder, where tamoxifen decreased ERα and PR expression, further stimulating ERß. In addition, tamoxifen-dosing decreased MetS-induced overexpression of inflammatory and tissue remodeling genes. In prostate, sex steroid receptors, pro-inflammatory and pro-fibrotic genes were upregulated in MetS. However, tamoxifen did not affect them and even increased COX-2. In hBPH cells, 17ß-estradiol increased IL-8 secretion, an effect blunted by co-treatment with GPER antagonist G15 but not by ER antagonist ICI 182,780, which further increased it. GPER agonist G1 dose-dependently (IC50 = 1.6 nM) induced IL-8 secretion. In vitro analysis demonstrated that GPER silencing reverted these stimulatory effects. CONCLUSIONS: GPER can be considered the main mediator of estrogen action in prostate, whereas in bladder the mechanism appears to rely on ERα, as indicated by in vivo experiments with tamoxifen dosing. Limiting the effects of the MetS-induced estrogen action via GPER could offer new perspectives in the management of BPH/LUTS, whereas tamoxifen dosing showed potential benefits in bladder.

Can baseball improve balance in blind subjects?

AIM: Baseball, one of the most popular sports in the world, is a fast-moving sport that requires various motor abilities. Baseball is played also by blind subjects that participate in many other sports. In this study, we evaluated the role of the Italian modified version of baseball for blind subjects on balance. METHODS: This modified version of baseball maintains the fast-moving characteristic ensuring the athlete safety. Forty total blind subjects were enrolled: 20 baseball athletes and 20 sedentary participants, as control. The balance was evaluated using the Fukuda Test and Tinetti Test, both in silence and in noise. RESULTS: This baseball game may help to improve the balance ability in blind subjects. The balance was significantly improved in blind athletes as compared with blind sedentary subjects. CONCLUSION: Given the peculiar characteristics of play, this modified version of baseball seems effective in improving various motor skills that, once transferred into daily activities, may significantly ameliorate the quality of life of blind subjects.

Immunomodulatory effects of BXL-01-0029, a less hypercalcemic vitamin D analogue, in human cardiomyocytes and T cells.

Current immunosuppressive protocols have reduced rejection occurrence in heart transplantation; nevertheless, management of heart transplant recipients is accompanied by major adverse effects, due to drug doses close to toxic range. In allograft rejection, characterized by T-helper 1 (Th1) cell-mediated response, the CXCL10-CXCR3 axis plays a pivotal role in triggering a self-promoting inflammatory loop. Indeed, CXCL10 intragraft production, required for initiation and development of graft failure, supports organ infiltration by Th1 cells. Thus, targeting the CXCL10-CXCR3 axis while avoiding generalized immunosuppression, may be of therapeutic significance. Based on preclinical evidence for immunoregulatory properties of vitamin D receptor agonists, we propose that a less hypercalcemic vitamin D analogue, BXL-01-0029, might have the potential to contribute to rejection management. We investigated the effect of BXL-01-0029 on CXCL10 secretion induced by proinflammatory stimuli, both in human isolated cardiomyocytes (Hfcm) and purified CD4+ T cells. Mycophenolic acid (MPA), the active agent of mycophenolate mofetil, was used for comparison. BXL-01-0029 inhibited IFNgamma and TNFalpha-induced CXCL10 secretion by Hfcm more potently than MPA, impairing cytokine synergy and pathways. BXL-01-0029 reduced also CXCL10 protein secretion and gene expression by CD4+ T cells. Furthermore, BXL-01-0029 did not exert any toxic effect onto both cell types, suggesting its possible use as a dose-reducing agent for conventional immunosuppressive drugs in clinical transplantation.

Elocalcitol inhibits inflammatory responses in human thyroid cells and T cells.

T-helper 1 (Th1) cell-mediated inflammatory responses predominate in the early pathogenesis of Graves' disease (GD), whereas Th2 cell-mediated immunity may play a role in later stages. The chemokine CXCL10 and its receptor CXCR3 are expressed in most thyroid glands of early GD patients. Circulating CXCL10 levels inversely correlate with disease duration; CXCL10 maximal expression also correlates with interferon (IFN)gamma levels in recent GD onset. Methimazole (MMI) reduces CXCL10 secretion by isolated thyrocytes, decreases serum CXCL10 levels, and promotes a transition from Th1 to Th2 dominance in patients in GD active phase. Vitamin D receptor agonists exhibit antiinflammatory properties and promote tolerance induction. We investigated the effects and the mechanism of action of a nonhypercalcemic vitamin D receptor agonist, elocalcitol (BXL-628), compared with MMI on CXCL10 secretion induced by proinflammatory cytokines. Furthermore, we studied the effects of both drugs on Th1, Th17, and Th2 cytokine secretion in CD4+ T cells. ELISA, cytometry, immunocytochemistry, Western blot, and quantitative real-time PCR were used for protein and gene analysis. In human thyrocytes, elocalcitol inhibited IFNgamma and TNFalpha-induced CXCL10 protein secretion more potently than MMI. Elocalcitol impaired both cytokine intracellular pathways, whereas MMI was effective only on the IFNgamma pathway. In CD4+ T cells, elocalcitol decreased Th1- and Th17-type cytokines, and promoted Th2-type cytokine secretion. Elocalcitol and MMI inhibited Th1 cytokine-mediated responses in thyrocytes and CD4+ T cells. In addition, elocalcitol promoted a shift toward a Th2 response. In conclusion, elocalcitol could represent a novel pharmacological tool in the treatment of autoimmune thyroid diseases.

The phosphodiesterase 5 inhibitor tadalafil regulates lipidic homeostasis in human skeletal muscle cell metabolism.

PURPOSE: Tadalafil seems to ameliorate insulin resistance and glucose homeostasis in humans. We have previously reported that tadalafil targets human skeletal muscle cells with an insulin (I)-like effect. We aim to evaluate in human fetal skeletal muscle cells after tadalafil or I: (i) expression profile of I-regulated genes dedicated to cellular energy control, glycolitic activity or microtubule formation/vesicle transport, as GLUT4, PPARγ, HK2, IRS-1, KIF1C, and KIFAP3; (ii) GLUT4, Flotillin-1, and Caveolin-1 localization, all proteins involved in energy-dependent cell trafficking; (iii) activation of I-targeted paths, as IRS-1, PKB/AKT, mTOR, P70/S6K. Free fatty acids intracellular level was measured. Sildenafil or a cGMP synthetic analog were used for comparison; PDE5 and PDE11 gene expression was evaluated in human fetal skeletal muscle cells. METHODS: RTq-PCR, PCR, western blot, free fatty acid assay commercial kit, and lipid stain non-fluorescent assay were used. RESULTS: Tadalafil upregulated I-targeted investigated genes with the same temporal pattern as I (GLUT4, PPARγ, and IRS-1 at 3 h; HK2, KIF1C, KIFAP3 at 12 h), re-localized GLUT4 in cell sites positively immune-decorated for Caveolin-1 and Flotillin-1, suggesting the involvement of lipid rafts, induced specific residue phosphorylation of IRS-1/AKT/mTOR complex in association with free fatty acid de novo synthesis. Sildenafil or GMP analog did not affect GLUT4 trafficking or free fatty acid levels. CONCLUSION: In human fetal skeletal muscle cells tadalafil likely favors energy storage by modulating lipid homeostasis via IRS-1-mediated mechanisms, involving activation of I-targeted genes and intracellular cascade related to metabolic control. Those data provide some biomolecular evidences explaining, in part, tadalafil-induced favorable control of human metabolism shown by clinical studies.

Methimazole inhibits CXC chemokine ligand 10 secretion in human thyrocytes.

CXC chemokine ligand 10 (CXCL10) plays a pivotal role in the self-perpetuation of the inflammatory processes in patients with autoimmune thyroid disease. Treatment with methimazole (MMI) reduces serum CXCL10 in patients with Graves' disease. In isolated human thyrocytes, tumor necrosis factor (TNF)alpha demonstrates a potent synergistic effect on interferon (IFN)gamma-induced CXCL10 secretion. We investigated the mechanism underlying the synergism between IFNgamma and TNFalpha and the effect of MMI on CXCL10 secretion in human thyrocytes. A peroxisome proliferator-activated receptor gamma agonist, rosiglitazone (RGZ), a known inhibitor of T helper 1 (Th1)-mediated responses, was also studied for comparison. Experiments were carried out in human thyrocytes isolated from internodular parenchyma of thyroid tissues derived from patients who had undergone surgery for multinodular goiter. ELISA was used to measure CXCL10 levels in culture supernatant. Flow cytometry was used to assess IFNgamma membrane receptor expression. Specific mRNA analysis was performed by Taqman real-time PCR. Immunofluorescence was performed to detect nuclear translocation of nuclear factor-kappaB (NF-kappaB). In human thyrocytes, the synergistic effect of TNFalpha with IFNgamma on CXCL10 secretion is due to the upregulation of IFNgamma receptor expression. MMI decreased cytokine-induced CXCL10 secretion by reducing TNFalpha-induced upregulation of the IFNgamma receptor. RGZ decreased the cytokine-induced CXCL10 secretion by impairing NF-kappaB translocation, without affecting IFNgamma receptor. MMI and RGZ targeted thyrocytes with the same pharmacological potency, likely acting throughout different mechanisms. Targeting T helper 1-mediated autoimmune thyroid disease with drugs that impair different intracellular pathways could be a novel pharmacological tool.

Fibroblast growth factor and endothelin-1 receptors mediate the response of human striatal precursor cells to hypoxia.

Fetal striatal transplantation has emerged as a new therapeutic strategy in Huntington's disease (HD). Hypoxia is one of the microenvironmental stress conditions to which fetal tissue is exposed as soon as it is isolated and transplanted into the diseased host brain. Mechanisms that support neuroblast survival and replenishment of damaged cells within the HD brain in the hypoxic condition have yet to be fully elucidated. This study is aimed at investigating the molecular pathways associated with the hypoxic condition in human fetal striatal neuroblasts (human striatal precursor (HSP) cells), using the hypoxia-mimetic agent cobalt chloride (CoCl2). We analyzed the effect of CoCl2 on HSP cell proliferation and on the expression of hypoxia-related proteins, such as hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF). Moreover, we evaluated fibroblast growth factor 2 (FGF2; 50ng/ml) and endothelin-1 (ET-1; 100nM) proliferative/survival effects in HSP cells in normoxic and hypoxic conditions. Dose-response experiments using increasing concentrations of CoCl2 (50-750µM) showed that the HSP cell growth was unaffected after 24h, while it increased at 48h, with the maximal effect observed at 400µM. In contrast, cell survival was impaired at 72h. Hypoxic conditions determined HIF-1α protein accumulation and increased gene and protein expression of VEGF, while FGF2 and ET-1 significantly stimulated HSP cell proliferation both in normoxic and hypoxic conditions, thus counteracting the apoptotic CoCl2 effect at 72h. The incubation with selective receptor (FGFR1, endothelin receptor A (ETA) and endothelin receptor B (ETB)) inhibitors abolished the FGF2 and ET-1 neuroprotective effect. In particular, ET-1 stimulated HSP cell survival through ETA in normoxic conditions and through ETB during hypoxia. Accordingly, ETA expression was down-regulated, while ETB expression was up-regulated by CoCl2 treatment. Overall, our results support the idea that HSP cells possess the machinery for their adaptation to hypoxic conditions and that neurotrophic factors, such as FGF2 and ET-1, may sustain neurogenesis and long-term survival through complex receptor-mediated mechanisms.

Expression and localization of VEGF receptors in human fetal skeletal tissues.

During development the vertebrate skeleton is the product of deriving cells from distinct embryonic lineages. The craniofacial skeleton is formed by migrating cranial neural crest cells, whereas the axial and limb skeletons are derived from mesodermal cells. The Vascular Endothelial Growth Factors (VEGFs) / receptors (VEGFRs) system plays an important role in angiogenesis, as well as osteogenesis, during bone development, growth, and remodeling, attracting endothelial cells and osteoclasts and stimulating osteoblast differentiation. Recent evidence has shown that during development VEGFR-3 is also expressed in neural and glial precursors of forebrain and cerebellum, as well as in the eye. In this study, we found that VEGFR-1, VEGFR-2 and VEGFR-3 are expressed in human bone both in fetal and adult life. The gene expression levels were significantly higher in fetal samples especially in mandibles. In addition, higher levels of VEGFR-3 in orofacial district were confirmed by western blotting analysis. We also observed that in fetal mandibular samples VEGFRs colocalized in several osteoblasts, osteoclasts and osteoprogenitor cells. Furthermore, some cells coexpressed VEGFR-3 and ET-1, a marker of neural crest cells. The results demonstrated different expression of VEGFRs in human mandibular and femoral bones which could be correlated to their different structure, function and development during organogenesis. VEGFR-3 might represent a specific signal for ectomesenchymal lineage differentiation during early human development.