MicroRNA-126 (miR-126) facilitates angiogenesis and regulates endothelial cell function. Recent data suggest that miR-126 can serve as a biomarker for vascular disease. Although endothelial cells are enriched for miR-126, platelets also contain miR-126. In this paper, we investigated the contribution of platelets to the pool of miR-126 in plasma of patients with type 2 diabetes (DM2) and how this is affected by aspirin. In vitro platelet activation resulted in the transfer of miR-126 from the platelet to the plasma compartment, which was prevented by aspirin. In vivo platelet activation, monitored in patients with DM2 by measuring soluble P-selectin, correlated directly with circulating levels of miR-126. The administration of aspirin resulted both in platelet inhibition and concomitantly reduced circulating levels of platelet-derived microRNAs including miR-126. Platelets are a major source of circulating miR-126. Consequently, in patho-physiological conditions associated with platelet activation, such as diabetes type 2, the administration of aspirin may lead to reduced levels of circulating miR-126. Thus, the use of platelet inhibitors should be taken into account when using plasma levels of miR-126 as a biomarker.

A hallmark of inflammatory diseases is the excessive recruitment and influx of monocytes to sites of tissue damage and their ensuing differentiation into macrophages. Numerous stimuli are known to induce transcriptional changes associated with macrophage phenotype, but posttranscriptional control of human macrophage differentiation is less well understood. Here we show that expression levels of the RNA-binding protein Quaking (QKI) are low in monocytes and early human atherosclerotic lesions, but are abundant in macrophages of advanced plaques. Depletion of QKI protein impairs monocyte adhesion, migration, differentiation into macrophages and foam cell formation in vitro and in vivo. RNA-seq and microarray analysis of human monocyte and macrophage transcriptomes, including those of a unique QKI haploinsufficient patient, reveal striking changes in QKI-dependent messenger RNA levels and splicing of RNA transcripts. The biological importance of these transcripts and requirement for QKI during differentiation illustrates a central role for QKI in posttranscriptionally guiding macrophage identity and function.

Recent advances in live cell imaging have provided a wealth of data on the dynamics of transcription factors. However, a consistent quantitative description of these dynamics, explaining how transcription factors find their target sequences in the vast amount of DNA inside the nucleus, is still lacking. In the present study, we have combined two quantitative imaging methods, single-molecule microscopy and fluorescence recovery after photobleaching, to determine the mobility pattern of the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR), two ligand-activated transcription factors. For dexamethasone-activated GR, both techniques showed that approximately half of the population is freely diffusing, while the remaining population is bound to DNA. Of this DNA-bound population about half the GRs appeared to be bound for short periods of time (∼0.7 s) and the other half for longer time periods (∼2.3 s). A similar pattern of mobility was seen for the MR activated by aldosterone. Inactive receptors (mutant or antagonist-bound receptors) show a decreased DNA binding frequency and duration, but also a higher mobility for the diffusing population. Likely, very brief (≤1 ms) interactions with DNA induced by the agonists underlie this difference in diffusion behavior. Surprisingly, different agonists also induce different mobilities of both receptors, presumably due to differences in ligand-induced conformational changes and receptor complex formation. In summary, our data provide a consistent quantitative model of the dynamics of GR and MR, indicating three types of interactions with DNA, which fit into a model in which frequent low-affinity DNA binding facilitates the search for high-affinity target sequences.

Proper regulation of endothelial cell-cell contacts is essential for physiological functioning of the endothelium. Interendothelial junctions are actively involved in the control of vascular leakage, leukocyte diapedesis and the initiation and progression of angiogenesis. We found that the RNA-binding protein quaking is highly expressed by endothelial cells and that its expression was augmented by prolonged culture under laminar flow and the transcription factor KLF2 binding to the promoter. Moreover, we demonstrated that quaking directly binds to the mRNA of VE-cadherin and β-catenin and can induce mRNA translation mediated by the 3′UTR of these genes. Reduced quaking levels attenuated VE-cadherin and β-catenin expression and endothelial barrier function in vitro and resulted in increased bradykinin-induced vascular leakage in vivo. Taken together, we report that quaking is essential in maintaining endothelial barrier function. Our results provide novel insight into the importance of post-transcriptional regulation in controlling vascular integrity.

Background. Treatment of inflammatory kidney diseases with systemic high-dose glucocorticoids (GCs) has severe side effects. Liposomal encapsulation could facilitate local delivery of GCs to the inflamed kidney, as liposomes encapsulate their payload until extravasation at sites of inflammation, potentially resulting in local bioactivity. Our aim was to evaluate the ability of liposomes to accumulate locally after renal ischaemia–reperfusion injury in the rat and to study its effect on macrophages. Methods.In vitro, human macrophages were incubated with fluorescent liposomes, liposomal prednisolone, prednisolone, empty liposomes or saline. Uptake was studied microscopically and treatment effect was assessed by interkeukin 6 (IL-6) enzyme-linked immunosorbent assay. The mechanism of action was evaluated by analysing GC receptor activation by microscopy and quantitative polymerase chain reaction (qPCR). In vivo, rats were subjected to ischaemia–reperfusion injury and were injected intravenously with fluorescent liposomes, liposomal prednisolone, prednisolone, empty liposomes or saline. Uptake was measured by the FLARE camera and the treatment effect by immunohistochemistry for myeloid cells and qPCR for inflammatory markers. Results.In vitro, macrophages internalized liposomes after 8 hours. Prednisolone or liposomal prednisolone treatment reduced IL-6 production and both compounds induced translocation of the GC receptor to the nucleus and upregulation of PER1 messenger RNA (mRNA), indicating a similar mechanism of action. In vivo, fluorescent liposomes accumulated in the inflamed kidney. Liposomal prednisolone treatment increased the presence of ED2-positive anti-inflammatory macrophages and both prednisolone and liposomal prednisolone reduced monocyte chemoattractant protein-1 (MCP-1) mRNA production, indicating a reduced pro-inflammatory profile in the kidney. Conclusions. Liposomal encapsulation is a promising strategy for local delivery of glucocorticoids to the inflamed kidney.