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

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 HDAC9 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. HDAC9 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.

Dependence of Proximal GC Boxes and Binding Transcription Factors in the Regulation of Basal and Valproic Acid-Induced Expression of t-PA.

Objective. Endothelial tissue-type plasminogen activator (t-PA) release is a pivotal response to protect the circulation from occluding thrombosis. We have shown that the t-PA gene is epigenetically regulated and greatly induced by the histone deacetylase (HDAC) inhibitor valproic acid (VPA). We now investigated involvement of known t-PA promoter regulatory elements and evaluated dependence of potential interacting transcription factors/cofactors. Methods. A reporter vector with an insert, separately mutated at either the t-PA promoter CRE or GC box II or GC box III elements, was transfected into HT-1080 and HUVECs and challenged with VPA. HUVECs were targeted with siRNA against histone acetyl transferases (HAT) and selected transcription factors from the Sp/KLF family. Results. An intact VPA-response was observed with CRE mutated constructs, whereas mutation of GC boxes II and III reduced the magnitude of the induction by 54 and 79% in HT-1080 and 49 and 50% in HUVECs, respectively. An attenuated induction of t-PA mRNA was observed after Sp2, Sp4, and KLF5 depletion. KLF2 and p300 (HAT) were identified as positive regulators of basal t-PA expression and Sp4 and KLF9 as repressors. Conclusion. VPA-induced t-PA expression is dependent on the proximal GC boxes in the t-PA promoter and may involve interactions with Sp2, Sp4, and KLF5.

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

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.

Histone deacetylase inhibitors stimulate tissue-type plasminogen activator production in vascular endothelial cells.

A reduced capacity for acute tissue-type plasminogen activator (t-PA) release is likely to be associated with an impaired endogenous defense against intravascular thrombosis. Efficient approaches to pharmacologically restore a defective t-PA release have been lacking, but recent observations suggest that histone deacetylase inhibitors (HDACis) enhance t-PA production in vitro. HDACis have diverse chemical structures and different HDAC-enzyme sub-class targeting. We here compared the effects of several clinically used HDACis on t-PA production in endothelial cells. Human umbilical vein endothelial cells were exposed to a panel of 11 different HDACis and t-PA mRNA and protein levels were quantified. All HDACis dose-dependently stimulated t-PA mRNA and protein expression with similar maximal efficacy but with different potencies. Already at low concentrations, the majority of inhibitors caused significant and sustained effects on t-PA production. In addition, selected HDACis were capable of normalizing t-PA production when suppressed by the inflammatory cytokine TNF-α. We conclude that HDACis targeting classical HDAC enzymes are powerful inducers of t-PA expression in cultured endothelial cells and could be promising candidates for pharmacological modulation of endogenous fibrinolysis in man.

Role of histone acetylation in the stimulatory effect of valproic acid on vascular endothelial tissue-type plasminogen activator expression.

AIMS: Stimulated release of tissue-type plasminogen activator (t-PA) is pivotal for an intravascular fibrinolytic response and protects the circulation from occluding thrombosis. Hence, an impaired t-PA production is associated with increased risk for atherothrombotic events. A pharmacological means to stimulate the production of this enzyme may thus be desirable. We investigated if the anti-epileptic drug valproic acid (VPA) is capable of enhancing t-PA expression in vitro in vascular endothelial cells, and further examined if its histone deacetylase (HDAC)-inhibitory activity is of importance for regulating t-PA expression. METHODS AND RESULTS: Human endothelial cells were exposed to valproic acid and t-PA mRNA and protein levels were quantified. Potential changes in histone acetylation status globally and at the t-PA promoter were examined by western blot and chromatin immunoprecipitation. Valproic acid dose-dependently stimulated t-PA mRNA and protein expression in endothelial cells reaching a 2-4-fold increase at clinically relevant concentrations and 10-fold increase at maximal concentrations. Transcription profiling analysis revealed that t-PA is selectively targeted by this agent. Augmented histone acetylation was detected at the t-PA transcription start site, and an attenuated VPA-response was observed with siRNA knock of HDAC3, HDAC5 and HDAC7. CONCLUSIONS: Valproic acid induces t-PA expression in cultured endothelial cells, and this is associated with increased histone acetylation at the t-PA promoter. Given the apparent potency of valproic acid in stimulating t-PA expression in vitro this substance may be a candidate for pharmacological modulation of endogenous fibrinolysis in man.

Hypothermia in cardiogenic shock reduces systemic t-PA release.

Therapeutic hypothermia has been found to improve hemodynamic and metabolic parameters in cardiogenic shock. Tissue plasminogen activator (t-PA) is a pro-thrombolytic enzyme, which also possesses pro-inflammatory properties. Interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α) are pro-inflammatory cytokines; interleukin 10 (IL-10) and transforming growth factor beta 1 (TGF-ß1) are anti-inflammatory cytokines. The aim of this experiment was to investigate the mechanism behind the protective effect of therapeutic hypothermia in cardiogenic shock. This was done by studying the effect of hypothermia on basal t-PA levels, peripheral t-PA release, and on the inflammatory response. Cardiogenic shock was induced by inflation of an angioplasty balloon in the proximal left anterior descending artery for 40 min in 16 pigs, followed by 110 min of reperfusion. The animals were randomized to hypothermia (33°C, n = 8), or normothermia (n = 8) at reperfusion. Hemodynamic parameters were continuously monitored. Plasma was sampled every 30 min for analysis of blood-gases and t-PA, and for analysis of inflammatory markers at baseline and at the end of the experiment. t-PA, IL-6 and TGF-ß1 increased during cardiogenic shock. Apart from favourably affecting hemodynamic and metabolic variables, hypothermia was found to reduce basal arterial and venous t-PA levels, and to inhibit the release of t-PA from the peripheral vascular bed. Hypothermia did not alter the inflammatory response. In conclusion, mild hypothermia improves hemodynamic and metabolic parameters in cardiogenic shock. This is associated with a reduction in basal t-PA levels and t-PA release from the peripheral vascular bed, but not with an altered inflammatory response.