Cultivation of the causative agent of human neoehrlichiosis from clinical isolates identifies vascular endothelium as a target of infection.

Candidatus (Ca.) Neoehrlichia mikurensis is the cause of neoehrlichiosis, an emerging tick-borne infectious disease characterized by fever and vascular events. The bacterium belongs to the Anaplasmataceae, a family of obligate intracellular pathogens, but has not previously been cultivated, and it is uncertain which cell types it infects. The goals of this study were to cultivate Ca. N. mikurensis in cell lines and to identify possible target cells for human infection. Blood components derived from infected patients were inoculated into cell lines of both tick and human origin. Bacterial growth in the cell cultures was monitored by real-time PCR and imaging flow cytometry. Ca. N. mikurensis was successfully propagated from the blood of immunocompromised neoehrlichiosis patients in two Ixodes spp. tick cell lines following incubation periods of 7-20 weeks. Human primary endothelial cells derived from skin microvasculature as well as pulmonary artery were also susceptible to infection with tick cell-derived bacteria. Finally, Ca. N. mikurensis was visualized within circulating endothelial cells of two neoehrlichiosis patients. To conclude, we report the first successful isolation and propagation of Ca. N. mikurensis from clinical isolates and identify human vascular endothelial cells as a target of infection.

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 inhibition enhances tissue plasminogen activator release capacity in atherosclerotic man.

UNLABELLED: The expression of the tissue plasminogen activator (t-PA) gene appears to be under epigenetic control and can be affected by histone deacetylation inhibition. The study aimed to test if histone deacetalyase inhibitor treatment lead to increased t-PA release or reduced exhaustion in t-PA release in response to stimulation, as well as change in plasminogen activator inhibitor-1 (PAI-1) in subjects with coronary disease. In this clinical study, 16 post-myocardial infarction subjects, the perfused forearm model was used with isoprenaline provocation during 20 minutes, to stimulate local t-PA release. Each subject was measured twice on the same day (repeated stimuli sequences) as well as on two different occasions, without treatment and after four weeks of treatment with valproic acid (500 mg, twice daily). Net forearm release for t-PA in response to isoprenaline at minutes 1.5, 3, 6, 9, 12, 15 and 18 was measured, allowing assessment of cumulative t-PA release. There was a reduction in the exhaustion of cumulative t-PA release during repeated and prolonged stimulation with valproic acid treatment compared to non-treatment. Plasma PAI-1 antigen was decreased following treatment compared to non-treatment (18.4 ± 10.0 vs. 11.0 ± 7.1 nanograms/ml respectively, mean with 95% confidence interval). These findings demonstrate that histone deacetylation inhibition increases the capacity for endogenous t-PA release in subjects with vascular disease. Furthermore, the fibrinolytic balance is favored with suppressed PAI-1 levels. More studies are needed to establish the clinical relevance of these findings. TRIAL REGISTRATION: EU Clinical Trials Register 2012-004950-27.

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.

Strenuous exercise increases late outgrowth endothelial cells in healthy subjects.

Endothelial progenitor cells (EPCs) and late outgrowth endothelial cells (OECs) seem to play an important role in vessel formation. While EPCs seem to exert their function mainly through a paracrine effect, the OECs can develop into mature endothelial cells and form tubular structures. Exercise is known to increase angiogenic factors that can mobilize EPCs; however, the effect on OECs is not known. We investigated the response to a single session of strenuous exercise on OECs, vascular endothelial growth factor (VEGF) and inflammatory cell levels in the healthy. Eleven healthy subjects performed 1 h of spinning exercise. Blood samples were collected at 1, 6, 24 and 48 h post-exercise for cell culture and biochemical analysis. OEC colonies doubled one hour after the spinning session (baseline 4.5 +/- 4.3 vs. 9.0 +/- 3.7, P < 0.05). Serum VEGF increased from 194 +/- 107 pg/ml at baseline to 224 +/- 111 pg/ml after 1 h, p = ns and neutrophilic granulocytes increased from 3.73 +/- 1.38 at baseline to 9.08 +/- 10.5 at 1 h (P < 0.01). The increased levels of OECs, VEGF and neutrophilic granulocytes declined gradually at the following time points. VEGF levels and neutrophilic granulocytes were highly correlated to OEC levels, r = 0.903 (VEGF) and r = 0.85 (neutrophilic granulocytes), respectively. Strenuous physical activity increases OEC colonies and is correlated to serum VEGF and neutrophilic granulocytes levels. An acute exercise-induced inflammatory response might be responsible for the VEGF release and subsequent increase of OECs. The clinical importance of these findings remains to be elucidated.

Suppression of endothelial t-PA expression by prolonged high laminar shear stress.

Primary hypertension is associated with an impaired capacity for acute release of endothelial tissue-type plasminogen activator (t-PA), which is an important local protective response to prevent thrombus extension. As hypertensive vascular remodeling potentially results in increased vascular wall shear stress, we investigated the impact of shear on regulation of t-PA. Cultured human endothelial cells were exposed to low (< or =1.5 dyn/cm(2)) or high (25 dyn/cm(2)) laminar shear stress for up to 48 h in two different experimental models. Using real-time RT-PCR and ELISA, shear stress was observed to time and magnitude-dependently suppress t-PA transcript and protein secretion to approximately 30% of basal levels. Mechanistic experiments revealed reduced nuclear protein binding to the t-PA specific CRE element (EMSA) and an almost completely abrogated shear response with pharmacologic JNK inhibition. We conclude that prolonged high laminar shear stress suppresses endothelial t-PA expression and may therefore contribute to the enhanced risk of arterial thrombosis in hypertensive disease.

Effects of IL-1beta 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.

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 x 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 (chi2 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 down regulated, and of shear stress-responsive genes, 133 genes were upregulated and 771 down regulated. 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.

Anti-inflammatory effects of budesonide in intestinal epithelial cells.

The glucocorticosteroid budesonide is one of the mainstay treatments in inflammatory bowel disease. The aim of this study was to investigate its effects on Caco-2 cells upon coculture with LPS stimulated macrophages, or with conditioned medium (CM) from the same. The mRNA expression of the chemokines IL-8 and ENA-78 were upregulated in Caco-2 cells, which was always more pronounced in the coculture, as compared to the CM situation, with the ENA-78 induction being higher than of IL-8. Addition of budesonide to the apical side of Caco-2 cells, decreased both IL-8 and ENA-78 mRNA levels in a dose dependent manner. A reduction in trans epithelial electrical resistance (TEER) and an increase in the apical-basolateral transport of fluoresceinated sulfonic acid (FS) were observed in the Caco-2 cells both in cocultures and CM experiments. The reduction in TEER was counteracted by budesonide, whereas it had no effect on the FS transport. We conclude that budesonide exert an anti-inflammatory effect directly on intestinal epithelial cells, which may contribute to the overall action of budesonide in the treatment of inflammatory bowel diseases.

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.