Summary of the unadjusted and adjusted Hazard Ratio for all-causes and cardiovascular mortality for all analysis.

<p>Summary of the unadjusted and adjusted Hazard Ratio for all-causes and cardiovascular mortality for all analysis.</p

Demographic characteristics of all patients (N = 37,981) with non-valvular atrial fibrillation and/or atrial flutter as hospitalization diagnosis from January 1, 1997 to December 31, 2012 identified in Danish nationwide administrative registries and treated with digoxin for these pathologies.

<p>Demographic characteristics of all patients (N = 37,981) with non-valvular atrial fibrillation and/or atrial flutter as hospitalization diagnosis from January 1, 1997 to December 31, 2012 identified in Danish nationwide administrative registries and treated with digoxin for these pathologies.</p

Survival curves.

<p>(a) Kaplan-Meier curve for all-cause mortality—main analysis–comparison among patients with and without chronic kidney disease. (b) Kaplan-Meier curve for all-cause mortality—first sensitivity analysis—comparison among patients with and without chronic kidney disease using a propensity-matched subpopulation. (c) Kaplan-Meier curve for all-cause mortality—second sensitivity analysis—comparison among patients with and without eGFR <30 ml/min/1.73m² using a propensity-matched subpopulation. (d) Cumulative incidence for cardiovascular mortality—main analysis–comparison among patients with and without chronic kidney disease (Chi-square test statistic: 28.263; p-value < 0.001). (e) Cumulative incidence for cardiovascular mortality—first sensitivity analysis—comparison among patients with and without chronic kidney disease using a propensity-matched subpopulation. (f) Cumulative incidence for cardiovascular mortality—second sensitivity—analysis comparison among patients with and without eGFR <30 ml/min/1.73m² using a propensity-matched subpopulation. Comparison among survival curves for all-causes was performed using log-rank test and the p-value was shown in the figure.</p

Flowchart.

<p>Flowchart of the study population and sensitivity analysis subpopulations. </p

Cell Surface Estrogen Receptor Alpha Is Upregulated during Subchronic Metabolic Stress and Inhibits Neuronal Cell Degeneration

<div><p>In addition to the classical nuclear estrogen receptor, the expression of non-nuclear estrogen receptors localized to the cell surface membrane (mER) has recently been demonstrated. Estrogen and its receptors have been implicated in the development or progression of numerous neurodegenerative disorders. Furthermore, the pathogenesis of these diseases has been associated with disturbances of two key cellular programs: apoptosis and autophagy. An excess of apoptosis or a defect in autophagy has been implicated in neurodegeneration. The aim of this study was to clarify the role of ER in determining neuronal cell fate and the possible implication of these receptors in regulating either apoptosis or autophagy. The human neuronal cell line SH-SY5Y and mouse neuronal cells in primary culture were thus exposed to chronic minimal peroxide treatment (CMP), a form of subcytotoxic minimal chronic stress previously that mimics multiple aspects of long-term cell stress and represents a limited molecular proxy for neurodegenerative processes. We actually found that either E2 or E2-bovine serum albumin construct (E2BSA, i.e. a non-permeant form of E2) was capable of modulating intracellular cell signals and regulating cell survival and death. In particular, under CMP, the up-regulation of mERα, but not mERβ, was associated with functional signals (ERK phosphorylation and p38 dephosphorylation) compatible with autophagic cytoprotection triggering and leading to cell survival. The mERα trafficking appeared to be independent of the microfilament system cytoskeletal network but was seemingly associated with microtubular apparatus network, i.e., to MAP2 molecular chaperone. Importantly, antioxidant treatments, administration of siRNA to ERα, or the presence of antagonist of ERα hindered these events. These results support that the surface expression of mERα plays a pivotal role in determining cell fate, and that ligand-induced activation of mER signalling exerts a powerful cell-survival signal. These results shed new light on the pathogenetic mechanisms leading to neuronal cell degeneration.</p> </div

Flow cytometry analysis of intracellular and cell surface ER expression in SH-SY5Y neuronal cells.

<p>Representative flow cytometry histogram plots show the fluorescence intensity of MC-20 and 1531 Abs for ERα and ERβ, respectively, compared with isotype controls, in (<b>A</b>) untreated cells (intracellular ER, left panels and mER, right panels), (<b>B</b>) CMP treated cells (intracellular ER), (<b>C</b>) CMP or CMP+NAC treated cells (mER). Dotted lines indicate isotype control staining and solid lines indicate anti-ERα- and anti-ERβ-labeled cells. Statistical differences between the peaks of cells were evaluated by the Kolmogorov-Smirnov test (a D/s_(n) ratio ≥15 was accepted as significant). (<b>D</b>) Values of mERα/isotype control mean fluorescence intensity (MFI) <i>ratio</i> are reported. The mean ± SD from 5 independent experiments is shown. Ctr, untreated cells.</p

Immunofluorescence analysis of cell surface mERα in SH-SY5Y cells.

<p>mERα expression (green fluorescence) in unpermeated SH-SY5Y cells and MAP2 expression (red fluorescence) in Triton X100 permeated cells: (<b>A</b>) untreated, (<b>B</b>) CMP or (<b>C</b>) NAC/CMP treated cells. Cells were counterstained with Hoechst dye to reveal nuclei (blue staining). Note the yellow spots suggesting a colocalization of ERα and MAP2 in CMP exposed cells. No yellow spots are instead observable in untreated and NAC/CMP treated cells. Magnification: 3500×.</p

Immunofluorescence analysis of cell surface mERα in rat hippocampal neurons.

<p>mERα expression (green fluorescence) in unpermeated hippocampal neuronal cells and MAP2 expression (red fluorescence) in Triton X100 permeated hippocampal neuronal cells: (<b>A</b>) untreated, (<b>B, C</b>) CMP treated or (<b>D</b>) NAC exposed before CMP treatment. Note the yellow spots in (<b>B</b>) and (<b>C</b>) suggesting a colocalization of ERα and MAP2 in soma (<b>B</b>) and dendritic protrusions (<b>C</b>) of CMP exposed cells. No yellow spots are instead observable in untreated and NAC/CMP exposed hippocampal cells. Cells were counterstained with Hoechst dye to reveal nuclei (blue staining). Magnifications: (<b>A</b>) (<b>B</b>) (<b>D</b>) 3500×; (<b>C</b>) 6000×.</p

Effect of E2BSA and E2 on the activation of ERK and p38 MAPK.

<p>(<b>A</b>) Western blot analysis of p-ERK1/2 in untreated and CMP treated SH-SY5Y cells in presence or absence of estrogens. To ensure the presence of equal amounts of protein, the membranes were re-probed with a mouse anti-β-actin monoclonal Ab. Data from one representative experiment out of 10 are shown (left panel). Densitometric analysis of p-ERK1/2 levels relative to β-actin is also shown (right panel). Values are expressed as means ± SD from 10 independent experiments. *, <i>P</i><0.01. (<b>B</b>) Western blot analysis of p-p38 in untreated and CMP treated neuronal cells in presence of absence of estrogens. Data from one representative experiment out of 10 are shown (left panel). Densitometric analysis of p-p38 levels relative to β-actin is also shown (right panel). Values are expressed as means ± SD from 10 independent experiments. *, <i>P</i><0.01. Ctr, untreated cells.</p

Effect of E2BSA and E2 on the modulation of apoptosis and autophagy.

<p>(<b>A</b>) Flow cytometry analysis of apoptosis in untreated and CMP treated SH-SY5Y cells in presence or absence of E2BSA, E2 and ERα antagonist MPP. Results obtained in a representative experiment are shown. Numbers in upper and bottom right quadrants of each plot refer to AV/PI double positive cells and to AV single positive cells, respectively. (<b>B</b>) Mean ± SD of the percentages of AV positive cells obtained in 10 independent experiments is reported. * <i>P</i><0.001. (<b>C</b>) Values of MFI <i>ratio</i> (Bcl-2/isotype control) are reported. The mean ± SD from 10 independent experiments is shown. * <i>P</i><0.01 for CMP treated cells <i>vs</i> all other conditions. Ctr, untreated cells. (<b>D</b>) Western blot analysis of LC3-II in untreated and CMP treated SH-SY5Y cells in presence or absence of E2 or E2BSA. Blots shown are representative of 10 independent experiments. (<b>E</b>) Densitometry analysis of LC3-II levels relative to β-actin is shown. Values are expressed as mean ± SD. * <i>P</i><0.01. Ctr, untreated cells. (<b>F</b>) Flow cytometry analysis of apoptosis in untreated and CMP treated SH-SY5Y cells in presence or absence of the autophagy inhibitor 3-MA and E2BSA. Mean ± SD of the percentages of AV positive cells obtained in 3 independent experiments is reported. (<b>G</b>) Western blot analysis of LC3-II in CMP treated SH-SY5Y cells in presence or absence of the autophagy inhibitor 3-MA and E2BSA. Blots shown are representative of 3 independent experiments.</p