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

ABSTRACTCandidatus (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

Role of Histone Acetylation in the Stimulatory Effect of Valproic Acid on Vascular Endothelial Tissue-Type Plasminogen Activator Expression

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Impairment of Endothelial Thromboprotective Function by Haemodynamic and Inflammatory Stress - Implications for hypertensive disease

The physiologically most important activator of intravascular fibrinolysis is tissue-type plasminogen activator (t-PA). The endothelium synthesizes and stores t-PA and regulated release of the enzyme is an important local protective response to prevent thrombus extension. Previous work by our group has shown that both patients with primary and secondary hypertension have a reduced capacity to release t-PA upon stimulation, a defect that is likely to contribute to the enhanced risk for arterial occlusion and tissue infarction in these subjects. The mechanism of this impairment is unclear although our experimental studies have indicated that it could be a direct effect of the elevated blood pressure. In order to investigate if the impairment could be reversed by lowering the blood pressure, we used the perfused-forearm model to examine hypertensive subjects for stimulated t-PA release before and after antihypertensive treatment. The findings show that the capacity for stimulated t- PA release can be significantly improved by blood pressure lowering. Treatment increased the amount of t-PA released and also improved the rapidity of the response. The changes were of similar magnitude regardless of treatment with the angiotensin converting enzyme inhibitor lisinopril or the calcium antagonist felodipine, suggesting that the improvement was related to the blood pressure effect per se. To examine the underlying mechanism of blood pressure-induced suppression of t-PA, we explored the potential involvement of the two main haemodynamic forces tensile stress and shear stress. Using in vitro biomechanical experimental models and cultured endothelial cells we observed suppressed t-PA gene expression and protein secretion in response to prolonged cyclic strain stimulation and a magnitude dependent suppression of t-PA transcript with prolonged laminar shear stress. Moreover, all reductions of t-PA were consistently followed by inductions of the main inhibitor of t-PA, plasminogen activator inhibitor type 1 (PAI-1). Further, as hypertension is often associated with a low-grade inflammation, we investigated the impact of the prototypic proinflammatory cytokine tumor necrosis factor-a (TNF-a) on t-PA expression. Prolonged stimulation of cultured endothelial cells was observed to suppress t-PA gene and protein expression. Mechanistic experiments with pharmacologic inhibitors showed that the inhibitory effect was nuclear factor-kappaB (NF-kappaB) and p38 mitogen-activated protein kinase (p38 MAPK) dependent and indicated that potential effector molecules might be the transcription factors NF-kappaB and CREB interacting with the t-PA kappaB and CRE promoter elements, respectively. In conclusion, these findings show that the impaired capacity to release t-PA in hypertensive subjects is directly related to the elevated blood pressure. Data from experimental studies indicate that this impaired fibrinolytic response could be an effect of an enhanced tensile, shear and inflammatory stress acting on the endothelium

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

Histone Deacetylase Inhibition Enhances Tissue Plasminogen Activator Release Capacity in Atherosclerotic Man

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 nano-grams/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

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

DNA methylation in t-PA enhancer, upstream promoter, and proximal promoter regions in primary, p.0, and p.4 HUVECs.

<p>(A) The methylation level in the t-PA enhancer in primary, passage 0, and passage 4 HUVECs from 11 subjects (ID 001–011) as determined by direct sequencing of bisulphite-treated PCR-amplified DNA. All the CpG dinucleotides in the core enhancer, stretching from -7,150 to -8,110, were included in the analysis. The most 3’ enhancer CpG site, -7,355, corresponds to the t-PA -7,351 C/T enhancer polymorphism (which, using updated genome assemblies, has been remapped to -7,355 relative the major TIS). (B) The methylation levels in the upstream promoter and promoter regions from four subjects (ID 001–004). The 9 CpG dinucleotides in the t-PA proximal promoter, stretching from TIS to -240, were analysed, along with the 7 CpG dinucleotides in the region immediately upstream of the promoter (from -240 to -760).</p

t-PA gene expression in primary and cultured HUVECs.

<p>Relative mRNA expression of tissue-type plasminogen activator (t-PA) in non-cultured HUVECs and p.0-4 HUVECs from the same subjects as determined by real-time RT-PCR. 7 of the 11 subjects from the methylation analysis (ID 005–011) were included also in this analysis (total n = 12). p<0,001 (one-way ANOVA).</p