AimsStimulated 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 ResultsHuman 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.ConclusionsValproic 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.
Background The endogenous fibrinolytic system has rarely been considered as a target to prevent thrombotic disease. Tissue-type plasminogen activator (t-PA) production is potently increased by histone deacetylase (HDAC) inhibitors in endothelial cells in vitro, but whether this translates into increased vascular t-PA production and an enhanced fibrinolytic capacity in vivo is unknown. Objectives To determine whether the HDAC inhibitor valproic acid (VPA) stimulates production of t-PA in the vasculature of mice, and whether VPA pretreatment affects fibrin deposition and clot formation after mechanical vessel injury. Methods Mice were injected with VPA twice daily for up to 5 days. t-PA mRNA, and antigen expression in the mouse aorta and the circulating levels of t-PA were determined. Fibrin and thrombus dynamics after mechanical vessel injury were monitored with intravital confocal microscopy. Potential effects of VPA on platelets and coagulation were investigated. Results and Conclusions We found that VPA treatment increased vascular t-PA production in vivo and, importantly, that VPA administration was associated with reduced fibrin accumulation and smaller thrombi in response to vascular injury, but still was not associated with an increased risk of bleeding. Furthermore, we observed that higher concentrations of VPA were required to stimulate t-PA production in the brain than in the vasculature. Thus, this study shows that VPA can be dosed to selectively manipulate the fibrinolytic system in the vascular compartment and reduce thrombus formation in vivo.
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.
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.
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