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.
In response to inflammatory cytokines and chemokines, monocytes differentiate into macrophages. Comprehensive analysis of gene expression regulation of neuronal guidance cue (NGC) ligands and ...receptors in the monocyte-to-macrophage differentiation process is not available yet. We performed transcriptome profiling in both human primary PBMCs/PBMC-derived macrophages and THP-1 cells/THP-1-macrophages using microarray or RNA sequencing methods. Pathway analysis showed that the axonal guidance pathway is significantly regulated upon monocyte differentiation. We confirmed NGC ligands and receptors which were consistently regulated, including SEMA4D, SEMA7A, NRP1, NRP2, PLXNA1 and PLXNA3. The involvement of RNA-binding protein quaking (QKI) in the regulation of NGC expression was investigated using monocytes and macrophages from a QKI haplo-insufficient patient and her healthy sibling. This revealed a positive correlation of SEMA7A expression with QKI expression. In silico analysis of 3′UTRs of NGCs proposed the competitive binding of QKI to proximal microRNA targeting sites as the mechanism of QKI-dependent regulation of SEMA7A. RNA immunoprecipitation confirmed an interaction of QKI with the 3′UTR of SEMA7A. Loss of SEMA7A resulted in monocyte differentiation towards a more anti-inflammatory macrophage. Taken together, the axonal guidance pathway is regulated during monocyte-to-macrophage differentiation, and the regulation is in line with the necessary functional adaption for the specialised role of macrophages.
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.
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.
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.
Liposomal encapsulation is a promising strategy for local delivery of glucocorticoids to the inflamed kidney.
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•Human macrophages produce and secrete factor H and FHL-1.•Production of these complement factors is regulated by IFN-γ and CD40L.•Macrophages preferentially express and secrete FHL-1 ...when compared to factor H.
Factor H is a pivotal complement regulatory protein that is preferentially produced by the liver and circulates in high concentrations in serum. There has been an increasing interest in the extrahepatic production of complement factors, including by cells of the immune system, since this contributes to non-canonical functions of local complement activation and regulation. Here we investigated the production and regulation of factor H and its splice variant factor H-like protein 1 (FHL-1) by human myeloid cells. As validation, we confirmed the predominant presence of intact factor H in serum, despite a strong but comparable mRNA expression of CFH and FHL1 in liver. Comparable levels of CFH and FHL1 were also observed in renal tissue, although a dominant staining for FHL-1 was shown within the proximal tubules. Human in vitro generated pro- and anti-inflammatory macrophages both expressed and produced factor H/FHL-1, but this was strongest in pro-inflammatory macrophages. Production was not affected by LPS activation, but was increased upon stimulation with IFN-γ or CD40L. Importantly, in both macrophage subsets mRNA expression of FHL1 was significantly higher than CFH. Moreover, production of FHL-1 protein could be confirmed using precipitation and immunoblotting of culture supernatants. These data identify macrophages as producers of factor H and FHL-1, thereby potentially contributing to local complement regulation at sites of inflammation.
Atherogenesis involves a complex interaction between immune cells and lipids, processes greatly influenced by the vascular smooth muscle cell (VSMC) phenotype. The DNA glycosylase NEIL3 has ...previously been shown to have a role in atherogenesis, though whether this is due to its ability to repair DNA damage or to other non-canonical functions is not yet clear. Hereby, we investigate the role of NEIL3 in atherogenesis, specifically in VSMC phenotypic modulation, which is critical in plaque formation and stability.
Chow diet-fed atherosclerosis-prone Apoe−/− mice deficient in Neil3, and NEIL3-abrogated human primary aortic VSMCs were characterized by qPCR, and immunohistochemical and enzymatic-based assays; moreover, single-cell RNA sequencing, mRNA sequencing, and proteomics were used to map the molecular effects of Neil3/NEIL3 deficiency in the aortic VSMC phenotype. Furthermore, BrdU-based proliferation assays and Western blot were performed to elucidate the involvement of the Akt signaling pathway in the transdifferentiation of aortic VSMCs lacking Neil3/NEIL3.
We show that Neil3 deficiency increases atherosclerotic plaque development without affecting systemic lipids. This observation was associated with a shift in VSMC phenotype towards a proliferating, lipid-accumulating and secretory macrophage-like cell phenotype, without changes in DNA damage. VSMC transdifferentiation in Neil3-deficient mice encompassed increased activity of the Akt signaling pathway, supported by cell experiments showing Akt-dependent proliferation in NEIL3-abrogated human primary aortic VSMCs.
Our findings show that Neil3 deficiency promotes atherosclerosis development through non-canonical mechanisms affecting VSMC phenotype involving activation of the Akt signaling pathway.
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•Apoe−/−/Neil3−/− mice develop more atherosclerosis as compared to Apoe−/− mice.•Apoe−/−/Neil3−/− mice display increased medial VSMC area and layer disorganization.•Increased aortic VSMC proliferation in Apoe−/−/Neil3−/− mice and NEIL3-deficient primary human VSMCs.•Neil3 deficiency increases aortic VSMC phenotypic transdifferentiation towards an atherosclerotic macrophage-like cells.•Neil3/NEIL3 deficiency-dependent VSMC proliferation involves activation of the Akt signaling pathway.
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.
Abstract only Background: Modulation of cellular function necessitates a versatile utilization of the transcriptome, thereby altering the proteome. These functional adaptations are ...post-transcriptionally guided by RNA-binding proteins (RBP), which confer cells with the capacity to rapidly respond to various stimuli and stressors. We recently discovered a pivotal role for the RBP Quaking (QKI) in directing the conversion of monocytes into macrophages, with in particular alternative splicing of pre-mRNAs critically impacting the acquisition of a pro-inflammatory phenotype. Computational analysis indicated that this differentiation process was dependent on dynamic mobilization of the actin cytoskeletal network, which prompted us to focus our attention on an exclusion event of exon 13 at the C-terminus of the actin-capping protein gamma-Adducin (ADD3). At present, the functional consequences of ADD3 isoform switching are unknown. Methods and Results: Stimulation of primary human monocytes with GM-CSF initially led to a decrease in ADD3 mRNA expression while ADD3 exon 13 usage was preserved. However, mRNA expression was restored upon differentiation towards the pro-inflammatory macrophage phenotype (t=3 days), while exon 13 inclusion strikingly dropped from 47.2% ± 10.5 to 7.7% ± 2.6 (p=0.0387, n=3). Having previously shown that QKI protein expression increases upon conversion to a macrophage, we sought to pinpoint whether QKI specifically regulates this ADD3 splicing event. Therefore, we mutated the QKI binding site (ACUAA → ACGAA) proximal to exon 13 using an ADD3 minigene. These studies revealed that disrupting the capacity for QKI to bind at this intronic sequence almost completely abolished exon 13 splicing (ACGAA: 94.9% ± 0.0025 inclusion vs. ACUAA: 2.0% ± 0.0009 inclusion; p<0.0001, n=3). Using predictive bioinformatics tools, exclusion of exon 13 was found to induce conformational changes in ADD3 protein structure. We subsequently utilized atomic force microscopy to study actomyosin dynamics and cell stiffness, pressure and tension in monocytes overexpressing the distinct ADD3 isoforms. Conclusions: QKI-mediated splicing of ADD3 triggers an isoform switch that impacts monocyte and macrophage function by altering the actin cytoskeleton.
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.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
In the pathophysiologic setting of acute and chronic kidney injury, the excessive activation and recruitment of blood-borne monocytes prompts their differentiation into inflammatory macrophages, a ...process that leads to progressive glomerulosclerosis and interstitial fibrosis. Importantly, this differentiation of monocytes into macrophages requires the meticulous coordination of gene expression at both the transcriptional and post-transcriptional level. The transcriptomes of these cells are ultimately determined by RNA-binding proteins such as QUAKING (QKI), that define their pre-mRNA splicing and mRNA transcript patterns. Using two mouse models, namely (1)
mice (
) and (2) the conditional deletion in the myeloid cell lineage using the lysozyme 2-Cre (
mice), we demonstrate that the abrogation of
expression in the myeloid cell lineage reduces macrophage infiltration following kidney injury induced by unilateral urethral obstruction (UUO). The
and
mice both showed significant diminished interstitial collagen deposition and fibrosis in the UUO-damaged kidney, as compared to wild-type littermates. We show that macrophages isolated from
mice are associated with defects in pre-mRNA splicing. Our findings demonstrate that reduced expression of the alternative splice regulator QKI in the cells of myeloid lineage attenuates renal interstitial fibrosis, suggesting that inhibition of this splice regulator may be of therapeutic value for certain kidney diseases.
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