Estradiol Stimulates Vasodilatory and Metabolic Pathways in Cultured Human Endothelial Cells

Vascular effects of estradiol are being investigated because there are controversies among clinical and experimental studies. DNA microarrays were used to investigate global gene expression patterns in cultured human umbilical vein endothelial cells (HUVEC) exposed to 1 nmol/L estradiol for 24 hours. When compared to control, 187 genes were identified as differentially expressed with 1.9-fold change threshold. Supervised principal component analysis and hierarchical cluster analysis revealed the differences between control and estradiol-treated samples. Physiological concentrations of estradiol are sufficient to elicit significant changes in HUVEC gene expression. Notch signaling, actin cytoskeleton signaling, pentose phosphate pathway, axonal guidance signaling and integrin signaling were the top-five canonical pathways significantly regulated by estrogen. A total of 26 regulatory networks were identified as estrogen responsive. Microarray data were confirmed by quantitative RT-PCR in cardiovascular meaning genes; cyclooxigenase (COX)1, dimethylarginine dimethylaminohydrolase (DDAH)2, phospholipase A2 group IV (PLA2G4) B, and 7-dehydrocholesterol reductase were up-regulated by estradiol in a dose-dependent and estrogen receptor-dependent way, whereas COX2, DDAH1 and PLA2G4A remained unaltered. Moreover, estradiol-induced COX1 gene expression resulted in increased COX1 protein content and enhanced prostacyclin production. DDAH2 protein content was also increased, which in turn decreased asymmetric dimethylarginine concentration and increased NO release. All stimulated effects of estradiol on gene and protein expression were estrogen receptor-dependent, since were abolished in the presence of the estrogen receptor antagonist ICI 182780. This study identifies new vascular mechanisms of action by which estradiol may contribute to a wide range of biological processes

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Expression of selected genes from the microarray under different ICI 182780 concentrations.

<p>Data are expressed as percentage of control values and are mean±SEM of 4–7 values corresponding to 2 different experiments.</p

Estradiol up-regulated DDAH2 protein expression results in decreased ADMA production and increased NO release mediated by ER.

<p>HUVEC were exposed to 1 nmol/L estradiol with or without 1 µmol/L ICI182780, and protein expression of (A) DDAH1 and (B) DDAH2, along with (C) ADMA levels and NO production, were measured as stated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008242#s2" target="_blank">Materials and methods</a>. Data are percentage of control values and are mean ± SEM of 9–12 values (4 different experiments). * p<0.05 or ** p<0.01 vs. control cells, and † p<0.05 vs. estradiol-alone treated cells.</p

Estrogen receptor alpha and beta protein expression in HUVEC.

<p>Cells were exposed to 1 nmol/L estradiol with or without 1 µmol/L ICI182780 for 24 hours, and protein expression of (A) ERα and (B) ERβ were measured as stated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008242#s2" target="_blank">Materials and methods</a>. A typical immunoblotting image and relative levels assessed by densitometry of bands of 66-kDa (ERα) or 56-kDa (ERβ) are presented. Data are percentage of control values and are mean ± SEM of 6 values (3 different experiments).</p

Genes that changed more than 1.9-fold with estradiol.

<p>Genes that changed more than 1.9-fold with estradiol.</p

Significant genes included in the top ten canonical pathways presented in Figure 3.

<p>Significant genes included in the top ten canonical pathways presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008242#pone-0008242-g003" target="_blank">Figure 3</a>.</p

Estradiol up-regulated COX1 protein expression results in increased prostacyclin production through ER.

<p>HUVEC were exposed to 1 nmol/L estradiol with or without 1 µmol/L ICI182780, and protein expression of (A) COX1 and (B) COX2 and prostacyclin production (C) were measured as stated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008242#s2" target="_blank">Materials and methods</a>. Data are percentage of control values and are mean ± SEM of 6–8 values (3–4 different experiments). * p<0.05 or ** p<0.01 vs. control cells, and † p<0.05 vs. estradiol-alone treated cells.</p

Top ten signaling and metabolic pathways regulated by estradiol.

<p>For the functional categorization of genes, Fischer's exact test was used to calculate a p value (shown as bars) determining the probability that each biological function assigned to the network is due to chance alone. The ratio (shown as squares) represents the number of differentially expressed genes in a given pathway divided by total number of genes that make up that canonical pathway.</p