Direct in vitro action of estrone on uterine and white adipose tissue in obesity.

The hypothesis whether estrone (E1) could exhibit a direct action at uterus and white adipose tissue (WAT), under obesity was tested. In uterine tissue of obese rats, E1 increased nitric oxide (NO) synthesis, and reduced reactive oxygen species (ROS) production. The anti-oxidative action of E1 was sustained under inflammatory stress or high glucose levels. ICI 182780 or G15 compounds were employed as ER or GPER antagonists respectively. The action of E1 on ROS release involved ER participation; instead GPER mediated the acute stimulation on NO production. The antioxidative effect depends on NO-ROS balance. NO synthase (NOS) blockage suppressed the reduction in ROS synthesis elicited by E1, effect mediated by cNOS and not by iNOS. On WAT explants, E1 reduced ROS and thiobarbituric acid reactive substances production, and diminished leptin release. In summary, the data provide evidence that, in uterus and WAT, E1 counteracts inflammatory and oxidative stress induced by obesity.

Magnetic Nanoplatform With Novel Potential for the Treatment of Bone Pathologies: Drug Loading and Biocompatibility on Blood and Bone Cells.

Magnetic iron oxide nanoparticles (MNPs) coated with citric acid (MG@CA) are proposed as raw materials for the treatment of bone diseases. Citric acid (CA) was selected as coating due to its role in the stabilization of apatite nanocrystals and as a signaling agent for osteoblast activation. Raloxifene (Ral), curcumine (Cur) and methylene blue (MB) were employed as model drugs as therapeutic agents for bone diseases. Characterization of raw and drug loaded nanosystems was conducted in order to elucidate the mechanisms governing interactions between therapeutics and the magnetic platform. Biocompatibility studies were performed on red blood cells (RBCs) from peripheral human blood. Cytotoxicity was evaluated on endothelial cells (ECs); and viability was studied for bone cells exposed at concentrations of 1, 10 and 100 [Formula: see text]/mL of the magnetic nano-platform. MG@CA exhibited proper physicochemical properties for the applications intended within this work. It presented satisfactory biocompatibility on peripheral red blood cells. Only doses of 100 [Formula: see text]/mL induced a decrease in metabolic activity of ECs and MC3T3-E1 cells. Drug adsorption efficiency was estimated as 62.0, 15.0 and 54.0 % for Ral, Cur and MB and drug loading capability of 12.0, 20.0 and 13.6%, respectively.

Vascular response to stress: Protective action of the bisphosphonate alendronate.

BACKGROUND: Since several additional actions of bone bisphosphonates have been proposed, we studied the effect of the bisphosphonate alendronate (ALN) on the vascular response to environmental stress. METHODS: Primary cultures of endothelial cells (EC) and vascular smooth muscle cells (VSMC) exposed to strained conditions were employed for experimental evaluation. After ALN treatment, cell migration, proliferation, and angiogenesis assays were performed. The participation of signal transduction pathways in the biochemical action of ALN was also assessed. RESULTS: In VSMC cultures, ALN counteracted the stimulation of cellular migration elicited by the proinflammatory agent lipopolysaccharide (LPS) or by high levels of calcium and phosphorus (osteogenic medium). Indeed, ALN reduced the increase of VSMC proliferation evoked by the stressors. When LPS and osteogenic medium were added simultaneously, the enhancement of cell proliferation dropped to control values in the presence of ALN. The mechanism of action of ALN involved the participation of nitric oxide synthase, mitogen-activated protein kinase (MAPK), and protein kinase C (PKC) signaling pathways. The study revealed that ALN exhibits a proangiogenic action. On EC, ALN enhanced vascular endothelial growth factor (VEGF) synthesis, and induced capillary-like tube formation in a VEGF-dependent manner. The presence of vascular stress conditions (LPS or osteogenic medium) did not modify the proangiogenic action elicited by ALN. CONCLUSION: The findings presented suggest an extra-bone biological action of ALN, which could contribute to the maintenance of vascular homeostasis avoiding cellular damage elicited by environmental stress.

ß-cyclodextrin coating: improving biocompatibility of magnetic nanocomposites for biomedical applications.

The role Beta-cyclodextrin (ßCD) on improving biocompatibility on healthy cellular and animal models was studied upon a formulation obtained from the development of a simple coating procedure. The obtained nanosystems were thoroughly characterized by FTIR, TGA, atomic absorption spectroscopy, dynamic light scattering and zeta potential, TEM/HR-TEM and magnetic properties. ßCD might interact with the magnetic core through hosting OA. It is feasible that the nanocomposite is formed by nanoparticles of MG@OA dispersed in a ßCD matrix. The evaluation of ßCD role on biocompatibility was performed on two healthy models. To this end, in vivo studies were carried out on Caenorhabditis elegans. Locomotion and progeny were evaluated after exposure animals to MG, MG@OA, and MG@OA-ßCD (10 to 500 µg/mL). The influence of ßCD on cytotoxicity was explored in vitro on healthy rat aortic endothelial cells, avoiding alteration in the results derived from the use of transformed cell lines. Biological studies demonstrated that ßCD attaching improves MG biocompatibility.

Regulatory effects of estetrol on the endothelial plasminogen pathway and endothelial cell migration.

BACKGROUND: Estetrol (E4) is a natural estrogen produced solely during human pregnancy. E4 is suitable for clinical use since it acts as a selective estrogen receptor modulator. In clinical trials E4 has been seen to have little or no effect on coagulation. Hence, it is interesting to investigate whether E4 alters endothelial-dependent fibrinolysis. OBJECTIVES: We studied the effects of E4 on the fibrinolytic system and whether this could influence the ability of endothelial cells to migrate. In addition, we compared the effects of E4 with those of 17ß-estradiol (E2). STUDY DESIGN: Human umbilical vein endothelial cells (HUVEC) were obtained from healthy women. Expression of plasminogen-activator inhibitor-1 (PAI-1), urokinase-type plasminogen activator (u-PA) and tissue plasminogen activator (t-PA) proteins was evaluated by Western blot analysis. Endothelial cell migration was studied by razor-scrape horizontal and multiwell insert systems assays. RESULTS: E4 increased the expression of t-PA, u-PA and PAI-1 in HUVEC, but less so than did equimolar amounts of E2. The effects of E4 on t-PA, u-PA and PAI-1 were mediated by the induction of the early-immediate genes c-Jun and c-Fos. E4 in combination with E2 antagonized the effects induced by pregnancy-like E2 concentrations but did not impair the effects of postmenopausal-like E2 levels. We also found that the increased synthesis of PAI-1, u-PA and t-PA induced by E2 and E4 is important for horizontal and three-dimensional migration of HUVEC. CONCLUSIONS: These results support the hypothesis that E4 acts as an endogenous selective estrogen receptor modulator (SERM), controlling the fibrinolytic system and endothelial cell migration.

Manipulation of Mg2+-Ca2+ Switch on the Development of Bone Mimetic Hydroxyapatite.

Ionic substitution can affect essential physicochemical properties leading to a specific biological behavior upon implantation. Therefore, it has been proposed as a tool to increase the biological efficiency of calcium phosphate based materials. In the following study, we have evaluated the contribution of an important cation in nature, Mg2+, into the structure of previously studied biocompatible and biodegradable hydroxyapatite (HA) nanorods and its subsequent effect on its chemical, morphology, and bone mimetic articulation. Mg2+-substituted HA samples were synthesized by an aqueous wet-chemical precipitation method, followed by an hydrothermal treatment involving a Mg2+ precursor that partially replace Ca2+ ions into HA crystal lattice; Mg2+ concentrations were modulated to obtain a nominal composition similar to that exists in calcified tissues. Hydrothermally synthesized Mg2+-substituted HA nanoparticles were characterized by X-ray powder diffraction, FT-NIR and EDX spectroscopies, field emission scanning and high resolution transmission electron microscopies (FE-SEM, H-TEM). Molecular modeling combining ab initio methods and power diffraction data were also performed. Results showed that Mg2+-substitution promoted the formation of calcium deficient HA (cdHA) where Mg2+ replacement is energetically favored at Ca(1) position in a limited and specific amount directing the additional Mg2+ toward the surface of the crystal. The control of Mg2+ incorporation into HA nanorods gave rise to a tailored crystallinity degree, cell parameters, morphology, surface hydration, solubility, and degradation properties in a dose-replacement dependent manner. The obtained materials show qualities that conjugated together to drive an optimal in vitro cellular viability, spreading, and proliferation confirming their biocompatibility. In addition, an improved adhesion of osteoblast was evidenced after Mg2+-Ca2+ substitution.

Magnetic nanoparticles for drug targeting: from design to insights into systemic toxicity. Preclinical evaluation of hematological, vascular and neurobehavioral toxicology.

A simple two-step drug encapsulation method was developed to obtain biocompatible magnetic nanocarriers for the potential targeted treatment of diverse diseases. The nanodevice consists of a magnetite core coated with chitosan (Chit@MNPs) as a platform for diclofenac (Dic) loading as a model drug (Dic-Chit@MNPs). Mechanistic and experimental conditions related to drug incorporation and quantification are further addressed. This multi-disciplinary study aims to elucidate the toxicological impact of the MNPs at hematological, vascular, neurological and behavioral levels. Blood compatibility assays revealed that MNPs did not affect either erythrosedimentation rates or erythrocyte integrity at the evaluated doses (1, 10 and 100 µg mL-1). A microscopic evaluation of blood smears indicated that MNPs did not induce morphological changes in blood cells. Platelet aggregation was not affected by MNPs either and just a slight diminution was observed with Dic-Chit@MNPs, an effect possibly due to diclofenac. The examined formulations did not exert cytotoxicity on rat aortic endothelial cells and no changes in cell viability or their capacity to synthesize NO were observed. Behavioral and functional nervous system parameters in a functional observational battery were assessed after a subacute treatment of mice with Chit@MNPs. The urine pools of the exposed group were decreased. Nephritis and an increased number of megakaryocytes in the spleen were observed in the histopathological studies. Sub-acute exposure to Chit@MNPs did not produce significant changes in the parameters used to evaluate neurobehavioral toxicity. The aspects focused on within this manuscript are relevant at the pre-clinical level providing new and novel knowledge concerning the biocompatibility of magnetic nanodevices for biomedical applications.

Influence of chitosan coating on magnetic nanoparticles in endothelial cells and acute tissue biodistribution.

Chitosan coating on magnetic nanoparticles (MNPs) was studied on biological systems as a first step toward the application in the biomedical field as drug-targeted nanosystems. Composition of MNPs consists of magnetite functionalized with oleic acid and coated with the biopolymer chitosan or glutaraldehyde-cross-linked chitosan. The influence of the biopolymeric coating has been evaluated by in vitro and in vivo assays on the effects of these MNPs on rat aortic endothelial cells (ECs) viability and on the random tissue distribution in mice. Results were correlated with the physicochemical properties of the nanoparticles. Nitric oxide (NO) production by ECs was determined, considering that endothelial NO represents one of the major markers of ECs function. Cell viability was studied by MTT assay. Different doses of the MNPs (1, 10 and 100 µg/mL) were assayed, revealing that MNPs coated with non-cross-linked chitosan for 6 and 24 h did not affect neither NO production nor cell viability. However, a significant decrease in cell viability was observed after 36 h treatment with the highest dose of this nanocarrier. It was also revealed that the presence and dose of glutaraldehyde in the MNPs structureimpact on the cytotoxicity. The study of the acute tissue distribution was performed acutely in mice after 24 h of an intraperitoneal injection of the MNPs and sub acutely, after 28 days of weekly administration. Both formulations greatly avoided the initial clearance by the reticuloendothelial system (RES) in liver. Biological properties found for N1 and N2 in the performed assays reveal that chitosan coating improves biocompatibility of MNPs turning these magnetic nanosystems as promising devices for targeted drug delivery.

Differential regulation of endothelium behavior by progesterone and medroxyprogesterone acetate.

Medroxyprogesterone acetate (MPA) is a synthetic progestin commonly used in hormone replacement therapy (HRT). The aim of this research was to study and compare the effect of progesterone (Pg) and MPA on the regulation of cellular events associated with vascular homeostasis and disease. Platelet adhesion to endothelial cells (ECs), nitric oxide (NO) production, and cell migration were studied using murine ECs in vitro exposed to the progestins. After 7 min of treatment, MPA significantly inhibited NO synthesis with respect to control values; meanwhile, Pg markedly increased vasoactive production. In senile ECs, the stimulatory action of Pg decreases; meanwhile, MPA maintained its ability to inhibit NO synthesis. The presence of RU486 antagonized the action of each steroid. When ECs were preincubated with PD98059 (MAPK inhibitor) or chelerythrine (protein kinase C (PKC) inhibitor) before Pg or MPA treatment, the former totally suppressed the steroid action, but the PKC antagonist did not affect NO production. In the presence of a PI3K inhibitor (LY294002), a partial reduction in Pg effect and a reversal of MPA action were detected. Using indomethacin, the contribution of the cyclooxygenase (COX) pathway was also detected. On platelet adhesion assays, Pg inhibited and MPA stimulated platelet adhesion to ECs. Under inflammatory conditions, Pg prevented platelet adhesion induced by lipopolysaccharide (LPS); meanwhile, MPA potentiated the stimulatory action of LPS. Finally, although both steroids enhanced migration of ECs, MPA exhibited a greater effect. In conclusion, the data presented in this research provide evidence of a differential regulation of vascular function by Pg and MPA.

Cellular actions of testosterone in vascular cells: mechanism independent of aromatization to estradiol.

In this work we investigated the role of testosterone on cellular processes involved in vascular disease, and whether these effects depend on its local conversion to estradiol. Cultures of rat aortic endothelial and smooth muscle cells in vitro treated with physiological concentrations of testosterone were employed. Testosterone rapidly increased endothelial nitric oxide production. To evaluate whether this non genomic action was dependent on testosterone aromatization we used an aromatase inhibitor. Anastrozole compound did not modify the fast increase in nitric oxide production elicited by testosterone. The hormonal effect was completely blocked by an androgen receptor antagonist (flutamide); meanwhile it wasn't modified by the presence of an estrogen receptor antagonist (ICI182780).The possibility of intracellular estradiol synthesis was ruled out when no differences were found in estradiol measurements performed in culture incubation medium from control and testosterone treated cells. The 5α-reductase inhibitor finasteride partially suppressed the enhancement in nitric oxide production, suggesting that the effect of testosterone was partially due to dihydrotestosterone conversion. Testosterone stimulated muscle cell proliferation independent of local conversion to estradiol. When cellular events that play key roles in vascular disease development were analyzed, testosterone prevented monocyte adhesion to endothelial cells induced by a proinflammatory stimulus (bacterial lipopolysaccharides), and prompted muscle cell migration in presence of a cell motility inducer. In summary, testosterone modulates vascular behavior through its direct action on vascular cells independent of aromatization to estradiol. The cellular actions exhibited by the steroid varied whether cells were under basal or inflammatory conditions.

Testosterone modulates platelet aggregation and endothelial cell growth through nitric oxide pathway.

The aim of the present study was to investigate the effect of testosterone on the modulation of cellular events associated with vascular homeostasis. In rat aortic strips, 5-20 min treatment with physiological concentrations of testosterone significantly increased nitric oxide (NO) production. The rapid action of the steroid was suppressed by the presence of an androgen receptor antagonist (flutamide). We obtained evidence that the enhancement in NO synthesis was dependent on the influx of calcium from extracellular medium, because in the presence of a calcium channel blocker (verapamil) the effect of testosterone was reduced. Using endothelial cell (EC) cultures, we demonstrated that androgen directly acts at the endothelial level. Chelerythrine or PD98059 compound completely suppressed the increase in NO production, suggesting that the mechanism of action of the steroid involves protein kinase C and mitogen-activated protein kinase pathways. It is known that endothelial NO released into the vascular lumen serves as an inhibitor of platelet activation and aggregation. We showed that testosterone inhibited platelet aggregation and this effect was dependent on endothelial NO synthesis. Indeed, the enhancement of NO production elicited by androgen was associated with EC growth. The steroid significantly increased DNA synthesis after 24 h of treatment, and this mitogenic action was blunted in the presence of NO synthase inhibitor N-nitro-l-arginine methyl ester. In summary, testosterone modulates vascular EC growth and platelet aggregation through its direct action on endothelial NO production.