Insight on the Intracrinology of Menopause: Androgen Production within the Human Vagina.

In this study, we investigated steroidogenic gene mRNA expression in human vaginas and verified the ability of human vagina smooth muscle cells (hvSMCs) to synthesize androgens from upstream precursor dehydroepiandrosterone (DHEA). As a readout for androgen receptor (AR) activation, we evaluated the mRNA expression of various androgen-dependent markers. hvSMCs were isolated from vagina tissues of women undergoing surgery for benign gynecological diseases. In these cells, we evaluated mRNA expression of several steroidogenic enzymes and sex steroid receptors using real time reverse transcription-polymerase chain reaction. Androgen production was quantified with liquid chromatography tandem-mass spectrometry (LC-MS/MS). In vaginal tissues, AR mRNA was significantly less expressed than estrogen receptor α, whereas in hvSMCs, its mRNA expression was higher than progestin and both estrogen receptors. In hvSMCs and in vaginal tissue, when compared to ovaries, the mRNA expression of proandrogenic steroidogenic enzymes (HSD3ß1/ß2, HSD17ß3/ß5), along with 5α-reductase isoforms and sulfotransferase, resulted as being more abundant. In addition, enzymes involved in androgen inactivation were less expressed than in the ovaries. The LC-MS/MS analysis revealed that, in hvSMCs, short-term DHEA supplementation increased Δ4-androstenedione levels in spent medium, while increasing testosterone and DHT secretion after longer incubation. Finally, androgenic signaling activation was evaluated through AR-dependent marker mRNA expression, after DHEA and T stimulation. This study confirmed that the human vagina is an androgen-target organ with the ability to synthesize androgens, thus providing support for the use of androgens for local symptoms of genitourinary syndrome in menopause.

Physical activity counteracts metabolic syndrome-induced hypogonadotropic hypogonadism and erectile dysfunction in the rabbit.

Metabolic syndrome (MetS) clusters cardiovascular and metabolic risk factors along with hypogonadism and erectile dysfunction. Lifestyle modifications including physical exercise (PhyEx) are well-known treatments for this condition. In this study, we analyzed the effect of PhyEx on hypothalamic-pituitary-testis axis and erectile function by use of an animal MetS model, previously established in rabbits fed a high-fat diet (HFD). Rabbits fed a regular diet (RD) were used as controls. A subset of both groups was trained on a treadmill. HFD rabbits showed typical MetS features, including HG (reduced T and LH) and impairment of erectile function. PhyEx in HFD rabbits completely restored plasma T and LH and the penile alterations. At testicular and hypothalamic levels, an HFD-induced inflammatory status was accompanied by reduced T synthesis and gonadotropin-releasing hormone (GnRH) immunopositivity, respectively. In the testis, PhyEx normalized HFD-related macrophage infiltration and increased the expression of steroidogenic enzymes and T synthesis. In the hypothalamus, PhyEx normalized HFD-induced gene expression changes related to inflammation and glucose metabolism, restored GnRH expression, particularly doubling mRNA levels, and regulated expression of molecules related to GnRH release (kisspeptin, dynorphin). Concerning MetS components, PhyEx significantly reduced circulating cholesterol and visceral fat. In multivariate analyses, cholesterol levels resulted as the main factor associated with MetS-related alterations in penile, testicular, and hypothalamic districts. In conclusion, our results show that PhyEx may rescue erectile function, exert anti-inflammatory effects on hypothalamus and testis, and increase LH levels and T production, thus supporting a primary role for lifestyle modification to combat MetS-associated hypogonadism and erectile dysfunction.

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.

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.

Differential Effects of Testosterone and Estradiol on Clitoral Function: An Experimental Study in Rats.

INTRODUCTION: Female sexual response is a complex phenomenon in which psychological, neurologic, and vascular mechanisms and hormonal factors interact. During the arousal phase, they cooperate to increase genital blood flow, thus inducing engorgement of the clitoris and lubrication of the vagina. Regulation of vascular and non-vascular smooth muscle tone is the crucial event in the erectile process. Preclinical studies have suggested that nitric oxide (NO) is the main vasodilator neurotransmitter modulating, through the second messenger cyclic guanosine monophosphate (cGMP), clitoral flow vessels. AIM: To investigate the effects of sexual steroid hormones on pro-erectile and relaxant (mediated by NO and cGMP) and anti-erectile and contractile (mediated by ras homolog gene family member A [RhoA] and Rho-associated protein kinase [ROCK]) mechanisms in the clitoris using a validated animal model of female ovariectomized Sprague-Dawley rats. METHODS: Subgroups of ovariectomized rats were treated with 17ß-estradiol, progesterone, testosterone, or testosterone and letrozole for 6 weeks. The experimental groups were compared with a control group of intact rats. MAIN OUTCOME MEASURES: Sex steroids plasma levels were assessed and in vitro contractility studies were carried out in order to investigate the effect of ovariectomy and in vivo treatments on clitoris smooth muscle activity. Smooth muscle cells (SMCs) from rat clitoral biopsies were isolated and characterized. RhoA activity was determined in SMCs cell cultures. RNA from tissues and cells was analyzed by quantitative real-time RT-PCR. RESULTS: Using real-time polymerase chain reaction, testosterone treatment upregulated the expression of NO-mediated pathway genes (endothelial and neuronal NO synthase, guanylate cyclase soluble subunit-α3, guanylate cyclase soluble subunit-ß3, cGMP-dependent protein kinase 1, and phosphodiesterase type 5). Conversely, estrogen replacement upregulated the expression of calcium-sensitizing RhoA-ROCK pathway genes. In vitro contractility studies were performed on phenylephrine pre-contracted clitoris strips. Ovariectomy resulted in a decreased responsiveness to Y-27632, a ROCK inhibitor, which was fully restored by 17ß-estradiol supplementation. To further examine the effect of 17ß-estradiol on the RhoA-ROCK pathway, smooth muscle cells were isolated from rat clitoris and their migration capacity was evaluated. CONCLUSION: Collectively, these data demonstrate that testosterone improves the relaxation of vascular smooth muscle cells through the NO-cGMP pathway, and that testosterone and 17ß-estradiol are necessary to maintain a functional contractile and relaxant machinery in the clitoris. This new concept might provide support for the concomitant use of estrogen and testosterone during the treatment of sexual arousal disorders related to hormonal imbalance or insufficiency.

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.

Laparoscopic approach for urgent abdominal surgery in patients with left ventricular assist devices.

Despite the fact that the rate of surgical complications mainly related to the necessary anticoagulation has been reported to be significant in patients supported by ventricular assist devices (VADs) who underwent noncardiac surgery, we report two cases showing that adequate peri-operative management of medical therapy and utilization of mini-invasive surgical approaches (i.e. laparoscopy) may limit the risks of morbidity, especially when surgery is required on an urgent basis.

Temperature management during off-pump coronary artery bypass graft surgery: a randomized clinical trial on the efficacy of a circulating water system versus a forced-air system.

OBJECTIVE: The aim of this study was to evaluate the performance of a new temperature management system specifically designed for cardiac surgery (Allon ThermoWrapping Thermoregulation System; MTRE Advanced Technologies Ltd, Or Akiva, Israel) using a circulating-water garment and to compare it with a conventional forced-air cover system in order to determine whether it could reduce the incidence of perioperative hypothermia during off-pump coronary artery bypass graft (OPCAB) surgery. DESIGN: Prospective, randomized. SETTING: University, tertiary care hospital. PARTICIPANTS: Thirty-one patients undergoing primary OPCAB surgery. INTERVENTIONS: Patients undergoing OPCAB surgery were randomized into the new thermoregulation system, Allon (study group, n = 15), and the standard forced-air system, Bair Hugger (Sterile Cardiac Access blanket Model 645; Augustine SA, Berne, Switzerland) (control group, n = 16). MEASUREMENTS AND MAIN RESULTS: Rectal temperature was recorded each 30 minutes during surgery and at intensive care unit arrival. Patients in the study group had higher temperatures than the control group at all time points, and the difference reached statistical significance after 2 hours of surgery. Moreover, fewer patients in the study group suffered perioperative hypothermia (defined as rectal temperature <36 degrees C) than the control group (2/15 patients (13.3%) in the study group v 13/16 (81.3%) in the control group [p = 0.0006]). No difference in other outcomes was noted. None of the patients died in the hospital. There were no adverse events reported. CONCLUSIONS: The circulating-water garment, Allon ThermoWrapping Thermoregulation System, maintained normothermia during OPCAB surgery better than forced-air systems, especially after the first 2 hours of surgery, and it was not associated with surgical field disturbance.

Phosphorylcholine coating may limit thrombin formation during high-risk cardiac surgery: a randomized controlled trial.

BACKGROUND: During cardiopulmonary bypass, blood contact with the large nonendothelial surfaces of the extracorporeal circuit induces activation and consumption of platelets and plasma coagulation factors. Phosphorylcholine (Pc) coating of oxygenators has been designed to improve surface biocompatibility. We evaluated the effects of a Pc-coated oxygenator on blood coagulation in patients undergoing high-risk open heart surgery and receiving tranexamic acid. METHODS: Thirty-nine patients undergoing reoperative valvular or combined procedures were randomized to the use of an oxygenator treated with Pc coating (Pc group) or of a standard oxygenator (control group). Platelet count, soluble CD40 ligand, fibrinogen, antithrombin, D-Dimer, prothrombin fragment 1.2 (F1.2), and free plasma hemoglobin levels were measured at baseline, at aortic unclamping, and at arrival in the intensive care unit. RESULTS: Postoperative bleeding, need for blood products, and clinical outcomes were similar in the two groups. At unclamping, F1.2, a marker of in vivo thrombin formation, increased to a greater extent in control patients than in Pc patients (p = 0.02), and in the latter group of patients was positively correlated with aortic cross-clamp times (r = 0.70). Relative to baseline values, the percent decrease in platelet count, fibrinogen, and antithrombin levels was not significantly different in Pc patients and in control patients after adjustment for multiple comparisons, but the percent decrease in platelet counts was negatively correlated with F1.2 levels in the entire series of patients (r = -0.62, p < 0.0001). All the evaluated parameters were similar in the two groups of patients at arrival in the intensive care unit. CONCLUSIONS: For patients undergoing high-risk open heart surgery and receiving tranexamic acid, a phosphorylcholine-coated oxygenator may reduce intraoperative thrombin formation and the associated consumption of platelets, fibrinogen, and antithrombin.

Off-pump coronary artery bypass grafting reduces postoperative neurologic complications.

OBJECTIVE: Complications occurring after coronary artery bypass graft (CABG) surgery, particularly neurologic damage, have been mainly correlated with the use of cardiopulmonary bypass (CPB). The aim of this work was to compare postoperative outcomes of patients undergoing CABG surgery, with or without the use of CPB, focusing on neurologic events. DESIGN: Observational study. SETTING: University tertiary care hospital. PARTICIPANTS: Two thousand seven hundred and forty consecutive patients who underwent CABG surgery in the period January 1998 to January 2003. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: For 738 patients, the operation was performed off-pump (OP group), and for 2002 patients CPB was used (CPB group). OP and CPB groups were compared with regard to preoperative status, anesthetic management, and postoperative outcomes, by means of univariate and multivariate analyses. Surgeons' propensity to operate off-pump was based on patients' age, renal conditions, and hemodynamics. Univariate and multivariate analyses showed that CPB was associated with a higher incidence of type I neurologic events compared with OP technique (2.1% versus 0.9%, odds ratio [OR]: 2.6, 95% confidence interval [CI], 1.2-5.9). A history of previous stroke (OR: 2.7, 95% CI, 1.2-5.9) and advanced age (OR: 1.06 per year, 95% CI, 1.02-1.09) were additional independent predictors of postoperative type I neurologic events. CONCLUSIONS: In the authors' experience, off-pump CABG surgery offers some benefits compared with CPB in respect to major neurologic complications.

A mathematical model of neonatal tidal liquid ventilation integrating airway mechanics and gas transfer phenomena.

Tidal liquid ventilation (TLV) was proposed as an alternative to conventional mechanical ventilation in the case of surfactant-deficiency diseases, particularly for very premature subjects. Although many experimental studies have been conducted up to now, the effects of variations in ventilatory settings, such as frequency and tidal volume, on blood arterialization and lung mechanics have not been studied quantitatively. We developed a mathematical model simulating the breathing processes occurring during neonatal TLV treatments. The model integrates the description of O2 and CO2 transport, from the trachea to pulmonary capillary blood and vice versa, with the description of fluid mechanics within the airways and the saccules (the alveoli precursors). Gas transfer is described with a mono-dimensional model, accounting for convective and diffusive transport through the airways, coupled with a 3-compartment model, simulating gas diffusion between saccules, plasma and red blood cells, and chemical reactions dependent on the concentrations of gases and related chemical species. Mechanic loads on airways are calculated by means of a lumped-parameters approach. The model calculates mechanical stress and gas exchange as a function of the ventilatory settings. The integration of these results sheds light on possible ventilation strategies to allow for optimal management of blood arterialization and lung mechanical load.

Volume controlled apparatus for neonatal tidal liquid ventilation.

Conventional gas ventilation is often unsuccessful for premature neonatal patients suffering from respiratory distress syndrome (RDS). For such patients, liquid ventilation (LV) with perfluorocarbon (PFC) liquids has been proposed. By eliminating the air-liquid interface in saccules (the premature gas exchange structures), where scarce or absent surfactant production exists, pulmonary instability is avoided, lung compliance is improved, and atelectatic saccules are recruited, ultimately lowering the saccular pressure. Tidal LV involves administrating a liquid tidal volume to the patient at each respiratory cycle, and therefore requires a dedicated circuital setup to deliver, withdraw, and refresh the PFC during the treatment. We have developed a prototype liquid breathing system (LBS). The apparatus comprises two subcircuits managed by a personal computer based control system. The ventilation subcircuit performs inspiration/expiration with two sets of peristaltic pumps. A system to evaluate the true inspired/expired volumes was devised that consists of two reservoirs equipped with pressure transducers measuring the hydraulic head of the fluid therein. Volume accuracy was +/- 0.3 ml. The refresh subcircuit properly processes the PFC by performing filtration (DFA, Pall, NY), oxygenation, CO2 scavenge, and heat exchange (SciMed 2500, Life Systems, MN). The new apparatus has been used in preliminary animal tests on five newborn mini pigs with induced acquired RDS. The PFC used was RM-101 (Miteni, Milano, Italy). The animals were successfully supported for 4 hours each. Mean arterial O2 pressure was 131.4 mm Hg (range 79.0-184.2), and mean arterial CO2 pressure was 64.8 mm Hg (range 60.0-73.4).