Erik Ulfhammer

Impaired Capacity for Stimulated Fibrinolysis in Primary Hypertension Is Restored by Antihypertensive Therapy

The increased risk for myocardial infarction and ischemic stroke in primary hypertension suggests that the condition is associated with prothrombotic mechanisms. We have shown that patients with hypertension have an impaired capacity for acute endothelial tissue-type plasminogen activator (t-PA) release, an important local protective response to prevent formation of intravascular thrombi. The aim of the present study was to investigate whether this impairment could be restored by the lowering of blood pressure. The capacity for acute t-PA release in response to intraarterial infusion of substance P at 8 pmol/min was investigated in a perfused-forearm study in 20 hypertensive patients (12 men and 8 women). Studies were performed when patients were untreated and after 8 weeks of randomized treatment with lisinopril or felodipine that lowered blood pressure by 26/10 and 24/12 mm Hg, respectively. The t-PA release response increased significantly with treatment (ANOVA, P=0.0001), with a similar effect in the 2 treatment groups. The peak release of t-PA increased from 257 (58) to 445 (77) ng/min×L/tissue−1 (t test, P=0.02). Also, treatment shortened the average time to peak secretion from 6.7 (1.4) to 2.7 (0.3) min (t test, P=0.01). In 6 patients with a delayed secretory peak (9 minutes or later), treatment normalized the response (&khgr;2 test, P=0.008). Antihypertensive therapy restores the capacity for acute t-PA release and improves the rapidity of the response in patients with primary hypertension. Similar responses with the 2 regimens suggest that the improvement is related to the blood pressure reduction as such. This effect may contribute to the thromboprotective effect of antihypertensive treatment.

Differential Global Gene Expression Response Patterns of Human Endothelium Exposed to Shear Stress and Intraluminal Pressure

We investigated the global gene expression response of endothelium exposed to shear stress and intraluminal pressure and tested the hypothesis that the two biomechanical forces induce a differential gene expression response pattern. Intact living human conduit vessels (umbilical veins) were exposed to normal or high intraluminal pressure, or to low or high shear stress in combination with a physiological level of the other force in a unique vascular ex vivo perfusion system. Gene expression profiling was performed by the Affymetrix microarray technology on endothelial cells isolated from stimulated vessels. Biomechanical forces were found to regulate a very large number of genes in the vascular endothelium. In this study, 1,825 genes were responsive to mechanical forces, which corresponds to 17% of the expressed genes. Among pressure-responsive genes, 647 genes were upregulated and 519 genes were downregulated, and of shear stress-responsive genes, 133 genes were upregulated and 771 downregulated. The fraction of genes that responded to both pressure and shear stimulation was surprisingly low, only 13% of the regulated genes. Our results indicate that the two different stimuli induce distinct gene expression response patterns, which can also be observed when studying functional groups. Considering the low number of overlapping genes, we suggest that the endothelial cells can distinguish between shear stress and pressure stimulation.

TNF-α mediated suppression of tissue type plasminogen activator expression in vascular endothelial cells is NF-κB- and p38 MAPK-dependent

Summary.  Background: Several proatherothrombotic conditions are associated with enhanced levels of circulating proinflammatory cytokines, which are believed to impair endothelial fibrinolytic capacity. Objective: This study aims at investigating how tumor necrosis factor (TNF)‐α regulates endothelial gene expression of the key fibrinolytic enzyme tissue‐type plasminogen activator (t‐PA). Methods: Cultured human umbilical vein endothelial cells were pretreated with selective inhibitors of the three major inflammatory signaling pathways activated by TNF‐α; the nuclear factor kappa‐B (NF‐κB), the p38 mitogen‐activated protein kinase (p38 MAPK), and the c‐jun N‐terminal kinase (JNK) pathways. Following TNF‐α stimulation, effects on t‐PA gene expression were evaluated with real‐time reverse transcriptase polymerase chain reaction and interactions of nuclear proteins with potential gene regulatory elements were studied with electrophoretic mobility shift assays. Results: Approximately 50% suppression of t‐PA gene expression was observed after prolonged stimulation with TNF‐α (≥24 h). The repression was shown to be preferentially dependent on NF‐κB activation, but also on p38 MAPK signaling. Further, we provide evidence for a TNF‐α induced binding of NF‐κB to the recently described κB site in the t‐PA gene and of cyclic adenosine monophosphate response element binding protein (CREB) to the t‐PA CRE‐like site. Conclusions: We conclude that TNF‐α impairs fibrinolytic capacity in vascular endothelial cells by a NF‐κB and p38 MAPK‐dependent suppression of t‐PA. This mechanism sheds a light on how inflammation contributes to the pathogenesis of cardiovascular diseases.

Influence of TNF-α and biomechanical stress on endothelial anti- and prothrombotic genes

Biomechanical stress modulates vascular tone, vascular remodelling and the spatial localisation of atherosclerotic plaques. Inflammatory cytokines, such as TNF-alpha, regulate expression of genes that impair the function of endothelial cells. This study investigates the combinatory effect of different biomechanical stresses and TNF-alpha on the expression of endothelial anti- and prothrombotic genes. Human umbilical vein endothelial cells were exposed to TNF-alpha and different levels of static/pulsatile tensile stress or shear stress. The response in endothelial cells to TNF-alpha was not modulated by tensile stress. However, shear stress was a more potent stimulus. Shear stress counteracted the cytokine-induced expression of VCAM-1, and the cytokine-suppressed expression of thrombomodulin and eNOS. Shear stress and TNF-alpha additively induced PAI-1, whereas shear stress blocked the cytokine effect on t-PA and u-PA. A flow profile characterized by high laminar shear stress seems to render the endothelial cell more resistant to inflammatory stress.

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.

Prolonged cyclic strain impairs the fibrinolytic system in cultured vascular endothelial cells.

Objective We previously reported that patients with primary hypertension have an impaired ability to release tissue-type plasminogen activator acutely from the vascular endothelium, and recently found that lowering blood pressure can restore this capacity. We hypothesized that the suppression of the fibrinolytic system is caused by the chronic pressure-induced increased haemodynamic load on the endothelium. Design and methods This study investigated the effect of the tensile force component of blood pressure by exposing cultured human aortic endothelial cells to 10% cyclic strain for 6–72 h. Messenger RNA levels of tissue-type plasminogen activator, urokinase-type plasminogen activator, and plasminogen activator inhibitor 1 were analysed using Taqman real-time reverse transcriptase–polymerase chain reaction and protein release by enzyme-linked immunosorbent assay. Results Tensile stimulation resulted in a transient initial upregulation of tissue-type plasminogen activator mRNA at 6 h (53%), which declined with time, and at 48 h had switched to a 28% downregulation. The reduction was sustained after 72 h. Tissue-type plasminogen activator protein secretion showed a similar but somewhat delayed response, with a transient increase in release at 6 h (60%), declining to a final 12% reduction at 72 h. A similar pattern was observed for urokinase-type plasminogen activator mRNA. By contrast, plasminogen activator inhibitor 1 mRNA expression and protein secretion increased at all timepoints (16–47%). Conclusion Prolonged tensile stimulation impairs fibrinolytic activity in human aortic endothelial cells by a dual action, with suppression of plasminogen activator expression and increased inhibitor production. This effect of tensile stress may contribute to the reduced fibrinolytic capacity observed in patients with hypertension.

A new biomechanical perfusion system for ex vivo study of small biological intact vessels.

The vascular endothelium transduces physical stimuli within the circulation into physiological responses, which influence vascular remodelling and tissue homeostasis. Therefore, a new computerized biomechanical ex vivo perfusion system was developed, in which small intact vessels can be perfused under well-defined biomechanical forces. The system enables monitoring and regulation of vessel lumen diameter, shear stress, mean pressure, variable pulsatile pressure and flow profile, and diastolic reversal flow. Vessel lumen measuring technique is based on detection of the amount of flourescein over a vessel segment. A combination of flow resistances, on/off switches, and capacitances creates a wide range of pulsatile pressures and flow profiles. Accuracy of the diameter measurement was evaluated. The diameters of umbilical arteries were measured and compared with direct ultrasonographic measurement of the vessel diameter. As part of the validation the pulsatile pressure waveform was altered, e.g., in terms of pulse pressure, frequency, diastolic shape, and diastolic reversal flow. In a series of simulation experiments, the hemodynamic homeostasis functions of the system were successfully challenged by generating a wide range of vascular diameters in artificial and intact human vessels. We conclude that the system presented may serve as a methodological and technical platform when performing advanced hemodynamic stimulation protocols.

Effect of shear stress, statins and TNF-α on hemostatic genes in human endothelial cells

Atherosclerotic plaque formation and progression are dependent on local shear stress patterns and inflammatory cytokines. Statins effectively reduce the progression of atherosclerosis and the incidence of cardiovascular events. However, the benefit of statins cannot be explained by cholesterol reduction alone. This study, investigated the non-lipid lowering effects of simvastatin and rosuvastatin on endothelial anti- and prothrombotic genes under different biomechanical and inflammatory stress conditions. Endothelial cells responded in a similar way to simvastatin and rosuvastatin. However, they were more sensitive to simvastatin. The statins had anti-inflammatory properties counteracting the TNF-α effect on the hemostatic genes studied. There was no observed synergistic effect between shear stress and simvastatin. Simvastatin had a counteracting effect on t-PA and PAI-1 compared to TNF-α and shear stress. Simvastatin blocked the TNF-α suppressive effect on thrombomodulin and eNOS, irrespective of shear stress. The strong inductive effect of TNF-α on VCAM-1 was counteracted by simvastatin and shear stress in an additive dose-response dependent way.

Effects of IL-1β and IL-6 on tissue-type plasminogen activator expression in vascular endothelial cells

INTRODUCTION The increased risk of thrombus formation in inflammatory conditions is generally considered to be due to the pro-coagulant effect of inflammatory cytokines. However, cytokines may also decrease the expression of the key fibrinolytic enzyme tissue-type plasminogen activator (t-PA) causing a reduced clearance of emerging intravascular thrombi. This study investigated the effects of the inflammatory cytokines interleukin (IL)-1beta and IL-6 on t-PA gene and protein expression, and elucidated by which signaling mechanisms the effects are mediated. MATERIALS AND METHODS Cultured human umbilical vein endothelial cells (HUVEC) were exposed to recombinant IL-1beta or IL-6. t-PA mRNA was quantified by real-time RT-PCR and t-PA antigen by ELISA. To clarify signaling mechanisms, selective inhibitors of major cytokine-activated signaling pathways were used. Interactions of nuclear proteins with potential t-PA gene regulatory elements were studied by gel shift assays. RESULTS Already at low concentrations, IL-1beta caused a distinct suppression of t-PA transcript and protein levels, mediated primarily by NF-kappaB signaling. This cytokine also increased binding of NF-kappaB subunits to a t-PA specific kappaB element. IL-6 stimulation per se did not affect t-PA mRNA or protein levels whereas soluble IL-6 receptor, in the presence of endogenous IL-6, suppressed t-PA expression. CONCLUSIONS We conclude that the proinflammatory cytokine IL-1beta impairs fibrinolytic capacity in vascular endothelial cells by an NF-kappaB dependent suppression of t-PA expression. In contrast, an effect of IL-6 on t-PA expression could not be detected, probably due to lack of IL-6 receptor expression on HUVEC.

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.