Endothelial function and the risk for endothelial dysfunction differ between males and females. Besides the action of estrogen, sex chromosome gene expression and programming effects also provoke ...this sexual dimorphism. MicroRNAs (miRNAs) have emerged as regulators of endothelial cell function and dysfunction. We here hypothesized distinct miRNA expression patterns in male versus female human endothelial cells that contribute to the functional differences. We used our well-established model of fetal endothelial cells isolated from placenta (fpEC) and analyzed sexual dimorphic miRNA expression and potentially affected biological functions. Next-generation miRNA sequencing of fpEC isolated after pregnancies with male and female neonates identified sex-dependent miRNA expression patterns. Potential biological pathways regulated by the altered set of miRNAs were determined using mirPath and mirSystem softwares, and suggested differences in barrier function and actin organization. The identified pathways were further investigated by monolayer impedance measurements (ECIS) and analysis of F-actin organization (Phalloidin). Nine miRNAs were differentially expressed in fpEC of male versus female neonates. Functional pathways most significantly regulated by these miRNAs included 'Adherens junction', 'ECM receptor interaction' and 'Focal adhesion'. These pathways control monolayer barrier function and may be paralleled by altered cytoskeletal organization. In fact, monolayer impedance was higher in fpEC of male progeny, and F-actin staining revealed more pronounced peripheral stress fibers in male versus female fpEC. Our data highlight that endothelial cell function differs between males and females already in utero, and that altered miRNAs are associated with sex dependent differences in barrier function and actin organization.
Osteosarcoma (OS) is the most common type of bone tumor, and has limited therapy options. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has striking anti-tumor effects in various tumors. Here, we ...investigated molecular mechanisms that mediate anti-tumor effects of 15d-PGJ2 in different OS cell lines. Human U2-OS and Saos-2 cells were treated with 15d-PGJ2 and cell survival was measured by MTT assay. Cell proliferation and motility were investigated by scratch assay, the tumorigenic capacity by colony forming assay. Intracellular ROS was estimated by H2DCFDA. Activation of MAPKs and cytoprotective proteins was detected by immunoblotting. Apoptosis was detected by immunoblotting and Annexin V/PI staining. The ex ovo CAM model was used to study growth capability of grafted 15d-PGJ2-treated OS cells, followed by immunohistochemistry with hematoxylin/eosin and Ki-67. 15d-PGJ2 substantially decreased cell viability, colony formation and wound closure capability of OS cells. Non-malignant human osteoblast was less affected by 15d-PGJ2. 15d-PGJ2 induced rapid intracellular ROS production and time-dependent activation of MAPKs (pERK1/2, pJNK and pp38). Tempol efficiently inhibited 15d-PGJ2-induced ERK1/2 activation, while N-acetylcystein and pyrrolidine dithiocarbamate were less effective. Early but weak activation of cytoprotective proteins was overrun by induction of apoptosis. A structural analogue, 9,10-dihydro-15d-PGJ2, did not show toxic effects in OS cells. In the CAM model, we grafted OS tumors with U2-OS, Saos-2 and MG-63 cells. 15d-PGJ2 treatment resulted in significant growth inhibition, diminished tumor tissue density, and reduced tumor cell proliferation for all cell lines. Our in vitro and CAM data suggest 15d-PGJ2 as a promising natural compound to interfere with OS tumor growth.
Osteosarcoma (OS) is the most common type of bone tumor, and has limited therapy options. 15-Deoxy-Δ
-prostaglandin J
(15d-PGJ
) has striking anti-tumor effects in various tumors. Here, we ...investigated molecular mechanisms that mediate anti-tumor effects of 15d-PGJ
in different OS cell lines. Human U2-OS and Saos-2 cells were treated with 15d-PGJ
and cell survival was measured by MTT assay. Cell proliferation and motility were investigated by scratch assay, the tumorigenic capacity by colony forming assay. Intracellular ROS was estimated by H
DCFDA. Activation of MAPKs and cytoprotective proteins was detected by immunoblotting. Apoptosis was detected by immunoblotting and Annexin V/PI staining. The ex ovo CAM model was used to study growth capability of grafted 15d-PGJ
-treated OS cells, followed by immunohistochemistry with hematoxylin/eosin and Ki-67. 15d-PGJ
substantially decreased cell viability, colony formation and wound closure capability of OS cells. Non-malignant human osteoblast was less affected by 15d-PGJ
. 15d-PGJ
induced rapid intracellular ROS production and time-dependent activation of MAPKs (pERK1/2, pJNK and pp38). Tempol efficiently inhibited 15d-PGJ
-induced ERK1/2 activation, while N-acetylcystein and pyrrolidine dithiocarbamate were less effective. Early but weak activation of cytoprotective proteins was overrun by induction of apoptosis. A structural analogue, 9,10-dihydro-15d-PGJ
, did not show toxic effects in OS cells. In the CAM model, we grafted OS tumors with U2-OS, Saos-2 and MG-63 cells. 15d-PGJ
treatment resulted in significant growth inhibition, diminished tumor tissue density, and reduced tumor cell proliferation for all cell lines. Our in vitro and CAM data suggest 15d-PGJ
as a promising natural compound to interfere with OS tumor growth.
Health behavior changes often require focusing on factors beyond the individual, particularly in low-income and underresourced areas. The purpose of this article was to assess associations between ...household structure and adult physical activity levels. Data were collected using Community Assessment for Public Health Emergency Response methodology to administer a household survey (n = 100). Household structure was calculated from summing the number of adults (⩾18 years) and children (<18 years) reported living in the house. Physical activity was measured using the International Physical Activity Questionnaire–Short Form. Adults living in households with two or more adults reported more MET (metabolic equivalent of task) minutes of physical activity per week than adults from households with only one adult. Adults living in households with two or more adults were twice as likely to meet aerobic guidelines for physical activity compared to adults living in households with only adult. Findings suggest the need for developing ecologic approaches in low-income communities to increase social support for physical activity in adults.
I(f) was shown to be present in adult human atrial and ventricular myocytes but data obtained from infant myocytes are lacking. We have studied I(f) in isolated ventricular myocytes from children ...undergoing surgical correction of tetralogy of Fallot (TOF; n = 5; mean age: 15.3 months). All recordings were made with the patch clamp technique in the whole cell mode at a temperature of 36-37 degrees C. A modified Tyrode solution containing 25 mM KCl was used to amplify I(f). Considering I(f) to be present when its current density at -120 mV was greater than 0.5 pA/pF, I(f) could be found in 28 out of 32 myocytes (88%). The mean current density was -2.01 +/- 0.3 pA/pF (mean +/- S.E.M.). First current activation occurred at -70 mV and I(f) could be reversibly inhibited by superfusing the myocytes with CsCl (2 mM). Half maximal activation (V(1/2)) of I(f) was at -80.3 +/- 1.0 mV (n = 28). Beta-adrenergic receptor stimulation with isoproterenol (1 microM) caused an acceleration of current activation and a shift of V(1/2) by 7.88 +/- 1.8 mV (n = 10) to less negative potentials. This study provides first evidence that the hyperpolarization-activated pacemaker current I(f) is present in infant human ventricular myocytes. Our results suggest that I(f) in ventricle of infants suffering from TOF has similar properties as I(f) in adult ventricle.
We have studied the inhibitory influence of the class III antiarrhythmic drug ambasilide (LU 47110) on the transient outward current Ito1 and the sustained current Iso following inactivation of Ito1, ...in human atrial myocytes. The two currents are separated by a mathematical procedure based on the amplitudes and time constants of the biexponential inactivation of the total outward current. The frequency dependence, the recovery from inactivation and the kinetics of activation and inactivation are described. Ambasilide reversibly and concentration dependently inhibited Ito1, Iso and the sodium current INa. Concentration required for half maximal inhibition (IC50) for the effects on Ito1 and Iso were 23.3 mumol/l and 45.7 mumol/l respectively, concentrations shown by others to be effective in terminating and preventing fibrillation in a dog atrial arrhythmia model. Ambasilide not only reduced the amplitude of Ito1 and Iso but also accelerated the time course of inactivation from 14.22 to 6.69 ms and from 202.3 to 87.9 ms respectively. The amplitude of Ito1 showed only a small dependence on stimulation frequency characteristic for human atrial myocytes, whereas Iso was reduced significantly at higher stimulation frequencies. Ambasilide did not change these relationships (0.1-4 Hz) and therefore did not show the reverse use-dependence known from other class III antiarrhythmic agents and which is an important property for a prospective antiarrhythmic drug. The lack of an effect of ambasilide on both steady-state activation and inactivation of Ito1, and the time constant of recovery from inactivation, suggests that ambasilide acts by changing conductance rather than by influencing the gating mechanism. The described characteristics make ambasilide an interesting substance in the group of class III antiarrhythmic drugs.
PDF
