L'Assunzione di associazioni estro-progestiniche seleziona due popolazioni femminili che differiscono in molti aspetti inclusi i processi di metilazione del DNA

Until now there has been little recruitment of women in clinical trials and this fact led to extrapolate data obtained in male gender also for women. The finding that gender differences are more numerous than one might think, suggests the importance of recruitment of women in the intervention studies. Moreover, the hormonal fluctuations that characterize woman's life (menstruation, pregnancy, lactation, menopause) and the use of oral contraceptives (CO) select different subsets of women. In this context we try to identify differences across a range of biomarkers blood, often used to measure the effectiveness of certain classes of drugs, by gender (males and females) and after stratification of the female population according the assumption of OC. For the current importance of epigenetic in the etiology of various diseases we analyze the degree of methylation of DNA in whole blood samples. We, also analyzed the behavior of macrophages (both in basal conditions and after stimulation with LPS), for their involvement in inflammatory processes in atherosclerosis in order to assess whether the administration of CO could affect their functionality. Our results confirm that gender differences exist between male and female but, more interesting, data evidence that female treated with OC behaved differently from untreated women versus some plasmatic parameters and in the degree of DNA methylation. All this shows that, in clinical trials of intervention, it is necessary to introduce different populations of women in view of the fact that 1/3 of women assumes OC, in the awareness to respect the complexity of the gender dimension in accordance with the recommendations of WHO and the European Commission

Cigarette smoking affects the differences between male and female phenotypes

Sex-gender medicine focuses on differences and similarities in health and disease between men and women. The present study focused on the existence of male and female phenotypes when routine demographic, biochemical and haematological data are considered and aimed to determine the influence of smoking on phenotypes and evaluate the role of body weight on sex-gender differences in view of the fact that some of them can be utilized as biomarkers of diagnosis, diseases and therapeutic response

LPS-stimulated human macrophages displayed sex differences in estrogen receptors α and β

Macrophages play a key role in immunity, inflammation, and atherosclerosis. Moreover, several evidences demonstrate that 17-β-estradiol (E2) plays a key role in inflammation and atherosclerosis through estrogen receptors (ERs) ERα and ERβ, processes that display sex differences. It has been largely demonstrated that male tissues express active ERs, but there is still lack of knowledge on their role in inflammation in males. Macrophages, which have ERα and ERβ, are a good model to evaluate the role of ER levels and activation in inflammation. The aim of our work was to evaluate the ability of lipopolysaccharide (LPS) to modulate, in a sex-specific way, the expression and the activation status of ERα and ERβ in blood monocytes-derived macrophages (MDMs) from healthy men and women. MDMs were isolated from blood of 7 adult men and 7 adult and fertile women (aged 21 - 35 years), and cultured. After 10 days of culture, MDMs were incubated with 100 ng / ml LPS for 24 h and lysed for the analysis of ERα, ERβ, P- ERα, p38 and P-p38 expression by Western Blotting. We found that in basal conditions, the expression of ERα and ERβ was significantly higher in female MDMs than in male ones. Importantly, LPS stimulation up-regulated ERα and ERα phosphorylation in both sexes, but this regulation was more pronounced in male MDMs. Moreover, LPS down-regulated ERβ level only in female MDMs. The expression of p38 and P-p38 proteins, used as marker of ERβ activity, did not display any sex differences. Finally, the ratios between ERα / ERβ and P-ERα / ERα were significantly higher in male than in female MDMs. Our findings show, for the first time, that LPS can modulate the activation of ERα but not that of ERβ, identifying a critical role of the subtype ERα in inflammatory responses mediated by LPS, at least in human MDMs. These results represent a starting point in understanding the influence of sex in the relationship between LPS and ERα

Gender differences in estrogenic compounds effect on human EPCs

Endothelial dysfunction has been defined as an “integrated risk factor”, and several gender difference have been reported in endothelial function. Several evidences suggest that endothelial progenitor cells (EPCs) are an important endogenous system that maintains integrity and vascular homeostasis. Their function is regulated by estrogens and estrogen receptors (ERs), but the effect of estrogenic compounds such as bisphenol A (BPA) and (R,R)-5,11-diethyl-5,6,11,12-tetrahydro-2,8-chrysenediol (THC) on human EPCs is unknown. BPA is used in plastic industry and BPA exposure is associated to abnormalities such as obesity, diabetes, disorders of the reproductive and immune systems, to endothelial dysfunction, and oxidative stress. This endocrine disruptor binds to ERα and ERβ with higher affinity for ERβ. THC is a specific agonist of ERα with a stronger ERβ antagonist activity. Therefore, the present work aimed to analyze if BPA and THC influence in a sex-specific manner the migration of human EPCs, an essential process in endothelial regeneration after vascular injury. EPCs were isolated from healthy adult men and women aged between 18 and 30 years, using a magnetic positive selection with the CD34 MicroBeads, a well-established marker of human progenitor cells. EPCs were also characterized for acetylated LDL Dil- (acLDL) and isothiocyanate (FITC)-conjugated with Ulex europaeus agglutinin I (lectin) uptake. The expression of ERα and ERβ was analysed by Western Blotting, while the migration assay was performed with the transwell chemotaxis assay. Male and female EPCs expressed both classical ERs: ERα was higher, but not significantly, in female cells, while ERβ was similarly expressed in both sexes. Male and female EPCs did not differ in basal migration. 17-β-estradiol (10-9 M e 10-10 M) significantly inhibited migration in female EPCs but not in male ones. Moreover, both 10-5 M THC and BPA (10-8 M) were able to bock migration only in female cells. Considering that BPA has a ERα and a prevalent ERβ agonist activity while THC has ERα agonistic activity and a prevalent ERβ antagonist activity, our data show that the effect on migration observed in female EPCs is mediated by ERα. Our data demonstrate that estrogenic compound have a sexual divergent effect on human EPCs, improving our knowledge on the gender differences observed in the pathophysiology of endothelial function

Sex differences in redox state, autophagy and lysosomal function

Modifications of the normal redox state are connected with numerous diseases and conditions such as cardiovascular diseases, diabetes mellitus and its complications, liver diseases, and aging which are characterized by numerous sex differences. Evidences suggest that redox state might be different in males and females. Autophagy is crucial for the maintaining cellular homeostasis process whereby cytoplasmic components are delivered to lysosomes for degradation. Alteration in constitutive autophagy is implicated in many pathological conditions, including heart diseases, diabetes mellitus and its complications, and liver diseases. A cross-talk between ROS and autophagy has been described. The current study was conducted to investigate the influence of sex on lipid and protein oxidation and autophagic response in the heart, the liver and the kidney obtained from young adult healthy male and female rats. 7 week old Sprague-Dawley rats were used to obtain heart, liver and kidneys. Malondialdehyde (MDA), and carbonylated proteins were measured by spectrophotometric methods for redox state assessment. The autophagy biomarkers Beclin-1, and microtubule-associated protein 1 light chain 3 (LC3), the mammalian target of rapamycin (mTOR; checkpoint in autophagic process), and the lysosomal associated membrane protein (LAMP-1; biomarker of lysosomes) were evaluated by Western blotting. Immunofluorescence analysis was also performed for LC3 and LAMP-1 colocalization. In the heart, Beclin-1, LC3-II/LC3-I were higher in males than in females suggesting that male heart have a major constitutive autophagy and this was linked with higher levels of carbonyl groups, indicating that protein oxidation could play a role. In the liver, it was found that LAMP-1 was higher in males and greatly colocalized with LC3 indicating a larger number of autophagolysosomes. None of the above parameters was significantly different in the kidneys of both sexes with the exception of MDA, which was significantly higher in females. These results suggest that the sex determinant affects the autophagy process and oxidation of proteins and lipids in an organ specific manner. It seems that the protein oxidation is more linked with constitutive autophagy, at least in cardiac ventricles, in comparison with lipid peroxidation

Autophagic process is sexually different in VSMCs from human male and female neonates

Sex has largely been neglected in cellular studies. Autophagy is a sophisticatedly reg- ulated homeostatic mechanism, which ensures cell’s constituent turnover. Under physiological conditions, autophagy levels are usually low (constitutive autophagy), but can be induced by numerous cellular stresses such as starvation. The influence of sex on autophagy has been studied either in vitro or in vivo, and previous our findings demonstrated the existence of sex differences in rat heart and HUVECs. Vascular smooth muscle cells (VSMCs) are a good experimental model for studying the physiopatholo- gy of the cardiovascular system, in which autophagy plays an important physiological role. Therefore, we investigated the occurrence of sexual dimorphism in constitutive and starvation-induced autophagy between the VSMCs obtained from human umbilical cord arteries (HUASMCs) of male (MHUASMCs) and female (FHUASMCs) neonates. HUASMCs were isolated from the umbilical cord of healthy and normal weight male and female neonates. The expression of oestrogen receptor (ER-α and ER-β) and the primary molecules involved in autophagic process [the mammalian target of rapamycin (mTOR), beclin-1 and microtubule-associated protein 1 light chain 3 (LC3)] were analysed by western blotting. Both cell types expressed both isoforms of the oestrogen receptor (ER): ER-β was higher expressed in the MHUASMCs than the FHUASMCs while ER-α was similarly expressed in both sexes. The level of constitutive autophagy, measured as LC3II/I ratio was higher in FHUASMCs than in MHUASMCs, while male cells had an higher expression of Beclin-1, indicating that constitutive autophagy was at least partly beclin-1-independent. mTOR activity, a regulator of autophagy, did not vary between the sexes, indicating that the observed differences could not be attributed to this central pathway. Starvation promoted autophagy in both MHUASMCs and FHUASMCs, but the increase was more pro- nounced in FHUASMCs. Our results show that sex-differences start in utero and are parameter-specific, suggesting that HUASMCs of both sexes are necessary in in vitro studies to elevate the quality and translational value of research results. The observed differences in the autophagic process could help to emeliorate our knowledge on sex-differences observed in cardiovascular diseases

Oral contraceptives modify DNA methylation and monocyte-derived macrophage function

Background: Fertile women may be encouraged to use contraception during clinical trials to avoid potential drug effects on fetuses. However, hormonal contraception interferes with pharmacokinetics and pharmacodynamics and modifies internal milieus. Macrophages depend on the milieu to which they are exposed. Therefore, we assessed whether macrophage function would be affected by the use of combined oral contraceptives (OCs) and if this influence depended on the androgenic or nonandrogenic properties of progestin. Methods: Healthy adult women were enrolled and stratified into two groups: women who did not use OCs (Fs) and women treated with OCs (FOCs). FOCs were further stratified as a function of androgenic (FOCA+) and non-androgenic (FOCA-) properties of progestins. Routine hematological, biochemical, inflammatory and endothelial dysfunction parameters were measured. Monocyte-derived macrophages (MDMs) were evaluated for the expression and activity of estrogen receptors and androgen receptors, and release of tumor necrosis factor α (TNFα) was measured from unstimulated and lipopolysaccharide-stimulated cells. Results: As is already known, the use of OCs changed numerous parameters: the number of lymphocytes, iron levels, total iron-binding capacity of transferrin, triglycerides, high-density lipoprotein, total cholesterol, and C-reactive protein increased, while prothrombin time and alkaline phosphatase decreased. Hormonal levels also varied: cortisol was higher in FOCs, while luteinizing hormone, folliclestimulating hormone, and testosterone were lower in FOCs. Asymmetric dimethylarginine, an index of endothelial function, was lower in FOC than in Fs, as were cysteine and bilirubin. The androgenic properties of progestins affected the activity of OCs: in particular, white blood cell count, hemoglobin, high-density lipoprotein and calcium were higher in FOCA- than in FOCA+, whereas percentage oxygen saturation and γ-glutamyl transpeptidase were lower in FOCA- than in FOCA+. Importantly, FOCs had a lower global DNA methylation, indicating that OC may have epigenetic effects on gene expression. OC did not modify the expression of androgen receptor but increased estrogen receptor α expression, more considerably in FOCA+, and decreased estrogen receptor β, more considerably in FOCA-. Importantly, the activation state of estrogen receptor β in FOCs was decreased, while estrogen receptor α was not active in either Fs or FOCs. Unstimulated MDMs obtained from FOCs showed higher release of TNFα in comparison with Fs. After lipopolysaccharide stimulation, the release of TNFα was significantly higher in Fs than in FOCs. Conclusions: OC use induced many changes in hematological and plasmatic markers, modifying hormonal levels, endothelial function, inflammation index and some redox state parameters, producing a perturbation of the internal milieu that impacted macrophagic function. In fact, different levels of estrogen receptor expression and release of TNFα were observed in macrophages derived from OC users. Some of the above activities were linked to the androgenic properties of progestin. Even though it is not known whether these effects are reversible, the results indicate that to avoid potential skewing of results only a single type of OC should be used during a single clinical trial.</br

Polimeri iper-ramificati a potenziale attività biologica

Nel corso di una ricerca volta all’individuazione di nuovi inibitori dell’HIV-1 integrasi (IN), sono stati sintetizzati e sottoposti a valutazione biologica l’acido 5,6-diidrossiindolo-2- carbossilico (DHICA), un intermedio nella biosintesi della melanina, ed una serie di suoi derivati. Questi composti erano stati progettati come analoghi conformazionalmente rigidi della porzione acrilica dell’ estere feniletilico dell’acido caffeico (CAPE). E’ risultato interessante il fatto che diversi derivati hanno mostrato attività inibitoria nei confronti dell’enzima HIV-1 IN a concentrazioni micromolari

Human umbilical endothelial cells (HUVECs) and sex differences

HUVECs are worldwide used to study the endothelial physiology and pathology that might be involved in sex and gender differences detected at the cardiovascular level. The present work characterised the phenotype of HUVECs in terms of morphology, proliferative and migratory capacity and in the gene expression of oestrogen and androgen receptors and nitric oxide synthase 3 (NOS3) to evaluated if they are sexually dimorphic. Moreover, autophagic process was analysed in male and female HUVECs (MHUVECs and FHUVECs), as autophagy is influenced by sex. Umbilical cords were obtained from healthy, normal weight, male and female neonates born to healthy non-obese and non-smoking women. HUVECs morphology was analysed by electron microscopy, and their function was investigated by proliferation, viability, wound healing and chemotaxis assays. Real-time PCR was used to evaluate gene expression for oestrogen and androgen receptors and for NOS3, while the expression of the primary molecules involved in autophagic process [(Akt, the mammalian target of rapamycin (mTOR), beclin-1 and microtubule-associated protein 1 light chain 3 (LC3)] and NOS3 were analysed by western blotting. FHUVECs showed significantly higher proliferation and migration rate, and NOS3 mRNA and protein expression than MHUVECs. Conversely, beclin-1 and the LC3-II/LC3-I ratio were higher in MHUVECs than in FHUVECs, indicating a higher autophagy in male cells as also indicated by ultrastructural analysis showing a buildup of autophagic vacuoles at different stages in MHUVECs. The expression of oestrogen and androgen receptor genes, the protein expression of Akt, mTOR, and cellular size and shape were not influenced by sex. Male and female neonates did not differ in body weight, but the weight of male babies was positively associated with the weight of the mother, suggesting that the weight of the mother may exert a different influence on male and female babies. Our findings indicate that sex differences exist from prenatal life and are parameter- specific, suggesting that a better quality of the research on the endothelium in vitro can be obtained by analyzing HUVECs of both sexes as well as its translational value. Moreover, the sex differences observed in HUVECs could help the diseases of adulthood because endothelial dysfunction has a key role in cardiovascular diseases, diabetes mellitus, neurodegeneration and immune diseases