Baseline characteristics of the study subjects (n = 10).

<p>Abbreviations: CRP =  C-reactive protein, LDL =  low density lipoprotein, HDL =  high density lipoprotein.</p><p>Baseline characteristics of the study subjects (n = 10).</p

Net forearm release rates of t-PA antigen during baseline and in response to 20 min infusion of Substance P (8 pmol/min) in untreated (▪) and VPA treated (◯) healthy subjects (n = 10).

<p>Baseline measurements 15 min before and 10 min after the infusion. 2-way ANOVA, mean and SEM.</p

Consort flow diagram.

<p>Consort flow diagram.</p

Study parameters.

<p>Study parameters.</p

Rapid and specific hypomethylation of enhancers in endothelial cells during adaptation to cell culturing

<p>Epigenetics, including DNA methylation, is one way for a cell to respond to the surrounding environment. Traditionally, DNA methylation has been perceived as a quite stable modification; however, lately, there have been reports of a more dynamic CpG methylation that can be affected by, for example, long-term culturing. We recently reported that methylation in the enhancer of the gene encoding the key fibrinolytic enzyme tissue-type plasminogen activator (t-PA) was rapidly erased during cell culturing. In the present study we used sub-culturing of human umbilical vein endothelial cells (HUVECs) as a model of environmental challenge to examine how fast genome-wide methylation changes can arise. To assess genome-wide DNA methylation, the Infinium HumanMethylation450 BeadChip was used on primary, passage 0, and passage 4 HUVECs. Almost 2% of the analyzed sites changed methylation status to passage 4, predominantly displaying hypomethylation. Sites annotated as enhancers were overrepresented among the differentially methylated sites (DMSs). We further showed that half of the corresponding genes concomitantly altered their expression, most of them increasing in expression. Interestingly, the stroke-related gene <i>HDAC9</i> increased its expression several hundredfold. This study reveals a rapid hypomethylation of CpG sites in enhancer elements during the early stages of cell culturing. As many methods for methylation analysis are biased toward CpG rich promoter regions, we suggest that such methods may not always be appropriate for the study of methylation dynamics. In addition, we found that significant changes in expression arose in genes with enhancer DMSs. <i>HDAC9</i> displayed the most prominent increase in expression, indicating, for the first time, that dynamic enhancer methylation may be central in regulating this important stroke-associated gene.</p

Dynamic Enhancer Methylation - A Previously Unrecognized Switch for Tissue-Type Plasminogen Activator Expression

<div><p>Tissue-type plasminogen activator (t-PA), which is synthesized in the endothelial cells lining the blood vessel walls, is a key player in the fibrinolytic system protecting the circulation against occluding thrombus formation. Although classical gene regulation has been quite extensively studied in order to understand the mechanisms behind t-PA regulation, epigenetics, including DNA methylation, still is a largely unexplored field. The aim of this study was to establish the methylation pattern in the t-PA promoter and enhancer in non-cultured compared to cultured human umbilical vein endothelial cells (HUVECs), and to simultaneously examine the level of t-PA gene expression. Bisulphite sequencing was used to evaluate the methylation status, and real-time RT-PCR to determine the gene expression level. While the t-PA promoter was stably unmethylated, we surprisingly observed a rapid reduction in the amount of methylation in the enhancer during cell culturing. This demethylation was in strong negative correlation with a pronounced (by a factor of approximately 25) increase in t-PA gene expression levels. In this study, we show that the methylation level in the t-PA enhancer appears to act as a previously unrecognized switch controlling t-PA expression. Our findings, which suggest that DNA methylation is quite dynamic, have implications also for the interpretation of cell culture experiments in general, as well as in a wider biological context.</p></div

Baseline venous and arterial plasminogen activator inhibitor-1 (PAI-1) antigen levels in untreated (▪) and VPA treated (grey square) subjects.

<p>*p<0.05, paired t-test, untreated versus treated.</p

Forearm blood flow during baseline and in response to 20 min infusion of Substance P (8 pmol/min) in untreated (▪) and VPA treated (◯) healthy subjects (n = 10).

<p>Baseline measurements 15 min before and 10 min after the infusion. 2-way ANOVA, mean and SEM.</p

Coronary t-PA-flux.

<p>Coronary t-PA fluxes are shown in Panel A for pre-ischemia (baseline), after 10 min of ischemia followed by 10 min of reperfusion for treated group (filled squares, n = 12) and for control group (open squares, n = 10). These are point measurements for observed t-PA fluxes at minutes 1, 3, 5, 7, and 10. One can note that there are different baselines for the two groups. The main result is derived from Panel A, and presented as the cumulative t-PA release over time in Panel B, as area under the curve (AUC). There is a clearly higher cumulative t-PA release for the VPA treated group (mixed between-within subjects ANOVA, p = 0.023); treated group (filled diamonds, n = 12) and control group (open diamonds, n = 10) data presented as mean ± SEM. There were also differences between groups (larger cumulative t-PA release in the treatment group) for specific measurements at minutes 10, 7, and 5 (t-test).</p

Absolute values of coronary lactate-fluxes.

<p>These are shown for pre-ischemia (baseline), after 10 min of ischemia followed by 10 min of reperfusion for treated group (filled squares, n = 12) and for control group (open squares, n = 10). Data are presented as mean ± SEM. Sampling of the ischemic area, as confirmed by positive lactate flux (regional myocardial lactate production in response to the local coronary occlusion), was observed in all animals (not shown individually) and is reflected by the immediate high positive lactate flux noted at reperfusion minute 1. This regional lactate flux was quickly ‘washed out’. No difference was found between groups (p = 0.853, mixed between-within subjects ANOVA).</p