Fam20C-mediated phosphorylation of osteopontin is critical for its secretion but dispensable for its action as a cytokine in the activation of hepatic stellate cells in liver fibrogenesis.

Osteopontin (OPN) is a phosphoglycoprotein secreted into the extracellular matrix upon liver injury, acting as a cytokine stimulates the deposition of fibrillary collagen in liver fibrogenesis. In livers of mice subjected to bile duct ligation (BDL) and in cultured activated hepatic stellate cells (HSCs), we show that OPN, besides being overexpressed, is substantially phosphorylated by family with sequence similarity 20, member C (Fam20C), formerly known as Golgi casein kinase (G-CK), which is exclusively resident in the Golgi apparatus. In both experimental models, Fam20C becomes overactive when associated with a 500-kDa multiprotein complex, as compared with the negligible activity in livers of sham-operated rats and in quiescent HSCs. Fam20C knockdown not only confirmed the role of Fam20C itself in OPN phosphorylation, but also revealed that phosphorylation was essential for OPN secretion. However, OPN acts as a fibrogenic factor independently of its phosphorylation state, as demonstrated by the increased expression of Collagen-I by HSCs incubated with either a phosphorylated or nonphosphorylated form of recombinant OPN. Collectively, our results confirm that OPN promotes liver fibrosis and highlight Fam20C as a novel factor driving this process by favoring OPN secretion from HSCs, opening new avenues for deciphering yet unidentified mechanisms underlying liver fibrogenesis.

Endometriosis Susceptibility to Dapsone-Hydroxylamine-Induced Alterations Can Be Prevented by Licorice Intake: In Vivo and In Vitro Study.

Endometriosis, an estrogen-dependent chronic gynecological disease, is characterized by a systemic inflammation that affects circulating red blood cells (RBC), by reducing anti-oxidant defenses. The aim of this study was to investigate the potential beneficial effects of licorice intake to protect RBCs from dapsone hydroxylamine (DDS-NHOH), a harmful metabolite of dapsone, commonly used in the treatment of many diseases. A control group (CG, n = 12) and a patient group (PG, n = 18) were treated with licorice extract (25 mg/day), for a week. Blood samples before (T0) and after (T1) treatment were analyzed for: i) band 3 tyrosine phosphorylation and high molecular weight aggregates; and ii) glutathionylation and carbonic anhydrase activity, in the presence or absence of adjunctive oxidative stress induced by DDS-NHOH. Results were correlated with plasma glycyrrhetinic acid (GA) concentrations, measured by HPLC-MS. Results showed that licorice intake decreased the level of DDS-NHOH-related oxidative alterations in RBCs, and the reduction was directly correlated with plasma GA concentration. In conclusion, in PG, the inability to counteract oxidative stress is a serious concern in the evaluation of therapeutic approaches. GA, by protecting RBC from oxidative assault, as in dapsone therapy, might be considered as a new potential tool for preventing further switching into severe endometriosis.

Human Sperm Capacitation Involves the Regulation of the Tyr-Phosphorylation Level of the Anion Exchanger 1 (AE1).

Bicarbonate uptake is one of the early steps of capacitation, but the identification of proteins regulating anion fluxes remains elusive. The aim of this study is to investigate the role of sperm solute carrier 4 (SLC4) A1 (spAE1) in the capacitation process. The expression, location, and tyrosine-phosphorylation (Tyr-P) level of spAE1 were assessed. Thereby, it was found that 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), an SLC4 family channel blocker, inhibited capacitation in a dose-dependent manner by decreasing acrosome reaction (ARC% 24.5 ± 3.3 vs 64.9 ± 4.3, p < 0.05) and increasing the percentage of not viable cells (NVC%), comparable to the inhibition by I-172, a cystic fibrosis transmembrane conductance regulator (CFTR) blocker (AR% 30.5 ± 4.4 and NVC% 18.6 ± 2.2). When used in combination, a synergistic inhibitory effect was observed with a remarkable increase of the percentage of NVC (45.3 ± 4.1, p < 0.001). spAE1 was identified in sperm membrane as a substrate for Tyr-protein kinases Lyn and Syk, which were identified as both soluble and membrane-bound pools. spAE1-Tyr-P level increased in the apical region of sperm under capacitating conditions and was negatively affected by I-172 or DIDS, and, to a far greater extent, by a combination of both. In conclusion, we demonstrated that spAE1 is expressed in sperm membranes and it is phosphorylated by Syk, but above all by Lyn on Tyr359, which are involved in sperm viability and capacitation.

Ameliorative effect of myo-inositol on red blood cell alterations in polycystic ovary syndrome: in vitro study.

Polycystic ovary syndrome (PCOS)is a gynecological endocrine disorder which is associated with systemic inflammatory status inducing red blood cells (RBC) membrane alterations related to insulin resistance and testosterone levels which could be greatly improved by myo-inositol (MYO) uptake. In this study we aim to evaluate the effect of MYO in reducing oxidative-related alterations through in vitro study on PCOS RBC. Blood samples from two groups of volunteers, control group (CG, n = 12) and PCOS patient group (PG, n = 12), were analyzed for band 3 tyrosine phosphorylation (Tyr-P), high molecular weight aggregate (HMWA), IgG in RBC membranes, and glutathione (GSH) in cytosol, following O/N incubation in the presence or absence of MYO. PCOS RBC underwent oxidative stress as indicated by higher band 3 Tyr-P and HMWA and increased membrane bound autologous IgG. Twenty four hours (but not shorter time) MYO incubation, significantly improved both Tyr-P level and HMWA formation and concomitant membrane IgG binding. However, no relevant modification of GSH content was detected. PCOS RBC membranes are characterized by increased oxidized level and enhanced sensitivity to oxidative injuries leading to potential premature RBC removal. MYO treatment is effective in reducing oxidative related abnormalities in PCOS patients probably restoring the inositol phospholipid pools of the membranes.

Astaxanthin Prevents Human Papillomavirus L1 Protein Binding in Human Sperm Membranes.

Astaxanthin (Asta), red pigment of the carotenoid family, is known for its anti-oxidant, anti-cancer, anti-diabetic, and anti-inflammatory properties. In this study, we evaluated the effects of Asta on isolated human sperm in the presence of human papillomavirus (HPV) 16 capsid protein, L1. Sperm, purified by gradient separation, were treated with HPV16-L1 in both a dose and time-dependent manner in the absence or presence of 30 min-Asta pre-incubation. Effects of HPV16-L1 alone after Asta pre-incubation were evaluated by rafts (CTB) and Lyn dislocation, Tyr-phosphorylation (Tyr-P) of the head, percentages of acrosome-reacted cells (ARC) and endogenous reactive oxygen species (ROS) generation. Sperm membranes were also analyzed for the HPV16-L1 content. Results show that HPV16-L1 drastically reduced membrane rearrangement with percentage of sperm showing head CTB and Lyn displacement decreasing from 72% to 15.8%, and from 63.1% to 13.9%, respectively. Accordingly, both Tyr-P of the head and ARC decreased from 68.4% to 10.2%, and from 65.7% to 14.6%, respectively. Asta pre-incubation prevented this drop and restored values of the percentage of ARC up to 40.8%. No alteration was found in either the ROS generation curve or sperm motility. In conclusion, Asta is able to preserve sperm by reducing the amount of HPV16-L1 bound onto membranes.

Persistent amenorrhea and decreased DHEAS to cortisol ratio after recovery from anorexia nervosa.

Persistent amenorrhea is a frequent condition affecting anorexic patients after stable weight recovery. It has been proposed that it could be due to alterations of the hypothalamic-pituitary-gonadal axis linked with persistent hormonal impairments, such as relative hypercortisolemia and hypoleptinemia, and psychological symptoms related to anorexia nervosa (AN). The aim of our study was to evaluate the metabolic and hormonal pattern involved in the persistence of amenorrhea after recovery from AN. Eight weight-recovered anorexic patients with amenorrhea were investigated and matched with 10 healthy eumenorrhoic women, comparable for age and BMI. Data showed basal FSH and LH values similar in both groups and a normal pituitaric response to LHRH administration. Morning serum cortisol was normal but significantly higher in patients, while dehydroepiandrosterone sulfate (DHEAS) to cortisol ratio, leptin and vitamin D were significantly lower in patients than controls. Women with previous AN presented insulin resistance and two patients showed an overall picture consistent with polycystic ovary syndrome (PCOS). In conclusion, long-lasting amenorrhea after recovery from AN is linked with a persistent hypothalamic dysfunction, although other concomitant causes like PCOS and insulin resistance should be considered. Decreased DHEAS to cortisol ratio is a new finding which could be correlated to the persistent hypogonadism.

Dapsone hydroxylamine-mediated alterations in human red blood cells from endometriotic patients.

Endometriosis, an estrogen-dependent chronic gynecological disease in women of reproductive age, is characterized by a systemic inflammation status involving also red blood cells (RBCs). In this study, we evaluated how the protein oxidative status could be involved in the worsening of RBC conditions due to dapsone intake in endometriotic women in potential treatment for skin or infection diseases. Blood samples from two groups of volunteers, control group (CG) and endometriosis patient group (PG), were analyzed for their content of band 3 tyrosine phosphorylation (Tyr-P) and high molecular weight aggregate (HMWA) in membranes, and glutathione (GSH) content and carbonic anhydrase (CA) activity in cytosol. In endometriotic patients, RBC showed the highest level of oxidative-related alterations both in membrane and cytosol. More interestingly, the addition of dapsone hydroxylamine (DDS-NHOH) could induce further increase of both membranes and cytosol markers, with an enhancement of CA activity reaching about 66% of the total cell enzyme amount. In conclusion, in PG the systemic inflammatory status leads to the inability of counteracting adjunctive oxidative stress, with a potential involvement of CA-related pathologies, such as glaucoma. Hence, the importance of the evaluation of therapeutic approaches worsening oxidative imbalance present in PG RBC is underlined.

Transient hypercortisolism and symptomatic hyperthyroidism associated to primary hyperparathyroidism in an elderly patient: case report and literature review.

BACKGROUND: Primary hyperparathyroidism (PHPT) is often found on routine blood tests, at a relatively asymptomatic stage. However many studies suggest different systemic effects related to PHPT, which could be enhanced by an abnormal cortisol release due to chronic stress of hyperparathyroidism. Being PHPT frequently found in the 6(th) to 7(th) decade of life, a careful and multifaceted approach should be taken. CASE PRESENTATION: We report the case of an elderly patient with symptomatic PHPT and incidental pulmonary embolism. He was treated with hydration, zoledronic acid, cinacalcet and high-dose unfractionated heparin. Parathyroid surgery was successfully performed, but patient's conditions suddenly worsened because of a transient thyrotoxicosis, probably induced by a previous exposure to iodine load and/or thyroid surgical manipulation. A short-term treatment with beta-blockers was introduced for symptomatic relief. The patient also presented a transient hypercortisolism with elevated ACTH, likely due to stress related not only to aging and hospitalization but also to PHPT, resolved only four months after parathyroid surgery. CONCLUSION: Chronic hyperparathyroidism has been linked with increased all-cause mortality. A functional chronic hypercortisolism could be established, enhancing PHPT related disorders. Only parathyroid surgery has been demonstrated to cure PHPT and complications related, showing similar outcome between older and younger patients. However, the management of post-operative period should be more careful in fragile patients. In particular, the early diagnosis and treatment of a transient post-operative thyrotoxicosis could improve recovery. Due to the increase in prevalence and the evidence of many related complications even in asymptomatic PHPT, expert opinion-based guidelines for surgical treatment of PHPT should be developed especially for elderly patients.

Astaxanthin Improves Human Sperm Capacitation by Inducing Lyn Displacement and Activation.

Astaxanthin (Asta), a photo-protective red pigment of the carotenoid family, is known for its multiple beneficial properties. In this study, the effects of Asta on isolated human sperm were evaluated. Capacitation involves a series of transformations to let sperm acquire the correct features for potential oocyte fertilization, including the generation of a controlled amount of reactive oxygen species (ROS), cholesterol depletion of the sperm outer membrane, and protein tyrosine phosphorylation (Tyr-P) process in the head region. Volunteers, with normal spermiogram values, were divided in two separate groups on the basis of their ability to generate the correct content of endogenous ROS. Both patient group (PG) and control group (CG) were analysed for Tyr-phosphorylation (Tyr-P) pattern and percentages of acrosome-reacted cells (ARC) and non-viable cells (NVC), in the presence or absence of Asta. In addition, the involvement of ROS on membrane reorganization and the presence of Lyn, a Src family kinase associated with lipid rafts, were investigated. Results show that Lyn is present in the membranes of human sperm, mainly confined in midpiece in resting conditions. Following capacitation, Lyn translocated to the head concomitantly with raft relocation, thus allowing the Tyr-P of head proteins. Asta succeeded to trigger Lyn translocation in PG sperm thus bypassing the impaired ROS-related mechanism for rafts and Lyn translocation. In this study, we showed an interdependence between ROS generation and lipid rafts and Lyn relocation leading the cells to undergo the successive acrosome reaction (AR). Asta, by ameliorating PG sperm functioning, may be utilised to decrease male idiopathic infertility.

Increased oxidation-related glutathionylation and carbonic anhydrase activity in endometriosis.

This study examined the possible involvement of carbonic anhydrase activation in response to an endometriosis-related increase in oxidative stress. Peripheral blood samples obtained from 27 healthy controls and 30 endometriosis patients, classified as having endometriosis by histological examination of surgical specimens, were analysed by multiple immunoassay and carbonic anhydrase activity assay. Red blood cells (RBC) were analysed for glutathionylated protein (GSSP) content in the membrane, total glutathione (GSH) in the cytosol and carbonic anhydrase concentration and activity. In association with a membrane increase of GSSP and a cytosolic decrease of GSH content in endometriosis patients, carbonic anhydrase significantly increased (P < 0.0001) both monomerization and activity compared with controls. This oxidation-induced activation of carbonic anhydrase was positively and significantly correlated with the GSH content of RBC (r = 0.9735, P < 0.001) and with the amount of the 30-kDa monomer of carbonic anhydrase (r = 0.9750, P < 0.001). Because carbonic anhydrase activation is implied in many physiological and biochemical processes linked to pathologies such as glaucoma, hypertension, obesity and infections, carbonic anhydrase activity should be closely monitored in endometriosis. These data open promising working perspectives for diagnosis and treatment of endometriosis and hopefully of other oxidative stress-related diseases. Endometriosis is a chronic disease associated with infertility and local inflammatory response, which is thought to spread rapidly throughout the body as a systemic subclinical inflammation. One of the causes in the pathogenesis/evolution of endometriosis is oxidative stress, which occurs when reactive oxygen species are produced faster than the endogenous antioxidant defence systems can neutralize them. Once produced, reactive oxygen species can alter the morphological and functional properties of endothelial cells, including permeability and adhesion molecule expression, thus contributing to ongoing inflammation. Due to their main cellular functions--delivery of O2 from lung to tissue and removal of CO2 from tissue to lung--red blood cells (RBC) are exposed to oxidative stress. Carbon dioxide in tissue capillaries diffuses into red cells, where it is rapidly hydrated by the action of cytosolic carbonic anhydrase. Analysis of the oxidation status of endometriotic RBC membranes showed a high content of glutathionylated proteins, indicating pre-existing oxidation-related alterations. The increase in glutathionylated proteins was correlated to increased carbonic anhydrase activity in endometriotic RBC compared with healthy controls. Carbonic anhydrase is a family of metalloenzymes involved in many physiological processes such as acid-base homeostasis, respiration, carbon dioxide and ion transport, and bone resorption, and in the regulation of ureagenesis, gluconeogenesis, lipogenesis and tumourigenesis. Due to the potential implication of carbonic anhydrase activation in many pathologies, such as glaucoma, hypertension, obesity and infections, carbonic anhydrase activity should be closely monitored in endometriosis to prevent possible complications and/or worsening of related conditions.

Effect of astaxanthin on human sperm capacitation.

In order to be able to fertilize oocytes, human sperm must undergo a series of morphological and structural alterations, known as capacitation. It has been shown that the production of endogenous sperm reactive oxygen species (ROS) plays a key role in causing cells to undergo a massive acrosome reaction (AR). Astaxanthin (Asta), a photo-protective red pigment belonging to the carotenoid family, is recognized as having anti-oxidant, anti-cancer, anti-diabetic and anti-inflammatory properties and is present in many dietary supplements. This study evaluates the effect of Asta in a capacitating buffer which induces low ROS production and low percentages of acrosome-reacted cells (ARC). Sperm cells were incubated in the presence or absence of increasing concentrations of Asta or diamide (Diam) and analyzed for their ROS production, Tyr-phosphorylation (Tyr-P) pattern and percentages of ARC and non-viable cells (NVC). Results show that Asta ameliorated both sperm head Tyr-P and ARC values without affecting the ROS generation curve, whereas Diam succeeded in enhancing the Tyr-P level but only of the flagellum without increasing ARC values. It is suggested that Asta can be inserted in the membrane and therefore create capacitation-like membrane alteration which allow Tyr-P of the head. Once this has occurred, AR can take place and involves a higher numbers of cells.

Long-Lasting Effects of Spironolactone after its Withdrawal in Patients with Hyperandrogenic Skin Disorders.

OBJECTIVE: Hyperandrogenic skin disorders, such as hirsutism, acne and alopecia, affect approximately 10-20% of women of reproductive age, reducing quality of life and causing psychological impairment. Spironolactone is a commonly used antiandrogen, especially in women who are not sexually active or have contraindications to hormonal contraceptives. The aim of this study was to evaluate the effects of spironolactone, especially after its withdrawal, in patients with hyperandrogenic skin disorders. METHODS: Retrospective analysis of 63 women with hyperandrogenic skin symptoms due to polycystic ovary syndrome (PCOS), treated with spironolactone for at least 6 months as first-line treatment. RESULTS: After a mean time of treatment of 25.7 months, all patients reported a significant improvement in hyperandrogenic skin disorders; only 5 patients were dissatisfied and required the addition of an oral contraceptive. The therapy was well tolerated and the most frequent side-effect was intermestrual bleeding in 68.2% of cases, affecting mainly classic PCOS phenotype. Thirthyeight patients showed prolonged effects 33.7 months after spironolactone withdrawal, whereas 20 relapsed 17.5 months after discontinuation. No significant difference in clinical and biochemical parameters was observed between these two groups both at baseline and after spironolactone treatment. Ovulatory PCOS patients were treated for a shorter time and reported earlier relapse than classic PCOS patients. CONCLUSION: Spironolactone is an effective and safe treatment for hyperandrogenic skin disorders, showing long-lasting effects even several months after its discontinuation.

Evaluation of correct endogenous reactive oxygen species content for human sperm capacitation and involvement of the NADPH oxidase system.

BACKGROUND: Generation of controlled amounts of reactive oxygen species (ROS) and phosphorylation of protein tyrosine residues (Tyr) are two closely related changes involved in sperm capacitation. This study investigated the effect of altered endogenous ROS production on Tyr-phosphorylation (Tyr-P), acrosome reaction (AR) and cell viability during sperm capacitation. The possible origin of the altered ROS production was also evaluated by apocynin (APO) or oligomycin (Oligo) addition. METHODS: A total of 63 samples of purified sperm were analysed for ROS production by enhanced chemiluminescence, Tyr-P pattern by immunocytochemistry, and AR and viability by fluorochrome fluorescein isothiocyanate (FITC)-labelled peanut (Arachis hypogaea) agglutinin and propidium iodide positivity, respectively. RESULTS: Samples were divided into four categories depending on the ability of sperm to produce ROS, expressed as Relative Luminescence Units (RLU), in capacitating conditions: low ROS production (LRP), range about 0.0-0.05 RLU; normal (NRP), 0.05-0.1 RLU; high (HRP), 0.1-0.4 RLU; very high (VHRP) 0.4-2.0 RLU. In NRP sperm heads, capacitation induced Tyr-P in 87.9 ± 4.3%, and the AR occurred in 62.5 ± 5.4% of cells; in LRP, HRP and VHRP Tyr-P labelling rarely spread over the head, acrosome-reacted cells only accounted for a small number of sperm, and the non-viable cells (NVC) were increased. The addition of APO, but not Oligo, drastically decreased ROS production in analysed samples. CONCLUSIONS: This study proposes the optimal threshold for endogenous ROS production for correct sperm viability and functioning, and indicates the direct involvement of APO-sensitive NADPH oxidase in ROS production.

Licorice: From Pseudohyperaldosteronism to Therapeutic Uses.

Licorice has been used as a medicinal plant from 2.500 years. It shows a wide range of biological and pharmacological activities, including anti-inflammatory and immune regulatory actions. One of its most known effects is the induction of hypertension, and it can induce what appears to be pseudohyperaldosteronism, due to glycyrrhetinic acid, the main active component of the root. Glycyrrhetinic acid and metabolites block the 11 beta-hydroxysteroid dehydrogenase type 2 and also bind mineralocorticoid receptors directly, acting as agonists. However, other interesting therapeutic uses of licorice are linked to its anti-androgen and estrogen-like activity, especially in the treatment of polycystic ovary syndrome (PCOS) in conjunction with spironolactone therapy. In this brief review, we report the main features and possible therapeutic uses of this ancient plant.

Aldosterone in Gynecology and Its Involvement on the Risk of Hypertension in Pregnancy.

Aldosterone is the main mineralocorticoid hormone, responsible of the regulation of fluid and electrolyte balance and blood pressure. It acts also as a pro-inflammatory factor responsible of an increased cardiovascular risk, independent from blood pressure values. After the discovery of mineralocorticoid receptor (MR) in mononuclear leukocytes, further studies supported its role in inflammatory and even autoimmune mechanisms underlying several diseases. In particular, recent studies reported a possible involvement of aldosterone in some gynecological conditions and diseases, characterized by inflammation, hypertension and increased cardio-metabolic risk, such as use of hormonal contraceptives, preeclampsia, polycystic ovary syndrome, uterine fibroids, and endometriosis. The aim of this mini-review is to report the possible involvement of aldosterone in all these gynecological conditions, suggesting different pathogenetic mechanisms and new target treatments of MR blockers for these diseases.

Mineralocorticoid receptor is involved in the aldosterone pathway in human red blood cells.

We have recently demonstrated that excessive aldosterone (Aldo) secretion in primary aldosteronism (PA) is associated with red blood cells (RBC) senescence. These alterations were prevented/inhibited by cortisol (Cort) or canrenone (Can) raising the hypothesis that Aldo effects in RBC may be mediated by mineralocorticoid receptor (MR), though to date MR has never been demonstrated in human RBC. The aim of this multicenter comparative study was to investigate whether Aldo effects were mediated by MR in these a-nucleated cells. We included 12 healthy controls (HC) and 22 patients with PA. MR presence and activation were evaluated in RBC cytosol by glycerol gradient sedimentation, Western blotting, immuno-precipitation and radioimmunoassay. We demonstrated that RBC contained cytosolic MR, aggregated with HSP90 and other proteins to form multiprotein complex. Aldo induced MR to release from the complex and to form MR dimers which were quickly proteolyzed. Cort induced MR release but not dimers formation while Can was not able to induce MR release. In addition, RBC cytosol from PA patients contained significantly higher amounts of both MR fragments (p<0.0001) and Aldo (p<0.0001) concentrations. In conclusion, in RBC a genomic-like Aldo pathway is proposed involving MR activation, dimerization and proteolysis, but lacking nuclear transcription. In addition, dimers proteolysis may ensure a sort of Aldo scavenging from circulation by entrapping Aldo in MR fragments.

Aldosterone receptor blockers spironolactone and canrenone: two multivalent drugs.

Canrenone is a derivative of spironolactone with lower antiandrogen activity. The drug is used only in few countries and can block all the side effects of aldosterone (ALDO). The drug is effective even in the presence of normal concentrations of ALDO. Mineralcorticoid receptor antagonists block the inflammatory activity of ALDO at the level of target tissues as heart, vessels and mononuclear leukocytes. Canrenone reduces the progression of insulin resistance and of microalbuminuria in type 2 diabetes and other related diseases. Both canrenone and hydrochlorothiazide can enhance the effect of treatment with ACE inhibitors and angiotensin II receptor blockers on microalbuminuria, but ALDO receptor blockers are more active. This different action is due to the fact that only canrenone blocks mineralocorticoid receptors. Serum potassium and renal function should be monitored before and during the treatment. ALDO receptor blockers are recommended in addition to polytherapy for resistant hypertension, but there are no studies on the effect of the drug as first-choice therapy.