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.
Abstract Background There's a large clinical need for novel vascular grafts. Tissue engineered blood vessels (TEBVs) have great potential to improve the outcome of vascular grafting procedures. Here, ...we present a novel approach to generate autologous TEBV in vivo . Polymer rods were engineered and implanted, evoking an inflammatory response that culminates in encapsulation by a fibrocellular capsule. We hypothesized that, after extrusion of the rod, the fibrocellular capsule differentiates into an adequate vascular conduit once grafted into the vasculature. Methods and results Rods were implanted subcutaneously in pigs. After 4 weeks, rods with tissue capsules grown around it were harvested. Tissue capsules were grafted bilaterally as carotid artery interposition. One and 4-week patency were evaluated by angiography whereupon pigs were sacrificed. Tissue capsules before and after grafting were evaluated on tissue remodeling using immunohistochemistry, RNA profiling and mechanical testing. Rods were encapsulated by thick, well-vascularized tissue capsules, composed of circumferentially aligned fibroblasts, collagen and few leukocytes, with adequate mechanical strength. Patency was 100% after 1 week and 87.5% after 4 weeks. After grafting, tissue capsules remodeled towards a vascular phenotype. Gene profiles of TEBVs gained more similarity with carotid artery. Wall thickness and αSMA-positive area significantly increased. Interestingly, a substantial portion of (myo)fibroblasts present before grafting expressed smooth muscle cell markers. While leukocytes were hardly present anymore, the lumen was largely covered with endothelial cells. Burst pressure remained stable after grafting. Conclusions Autologous TEBVs were created in vivo with sufficient mechanical strength enabling vascular grafting. Grafts differentiated towards a vascular phenotype upon grafting.
Atherosclerosis is the underlying pathology in a major part of cardiovascular disease, the leading cause of mortality in developed countries. The infiltration of monocytes into the vessel walls of ...large arteries is a key denominator of atherogenesis, making monocytes accountable for the development of atherosclerosis. With the development of high-throughput transcriptome profiling platforms and cytometric methods for circulating cells, it is now feasible to study in-depth the predicted functional change of circulating monocytes reflected by changes of gene expression in certain pathways and correlate the changes to disease outcome. Neuroimmune guidance cues comprise a group of circulating- and cell membrane-associated signaling proteins that are progressively involved in monocyte functions. Here, we employed the CIRCULATING CELLS study cohort to classify cardiovascular disease patients and healthy individuals in relation to their expression of neuroimmune guidance cues in circulating monocytes. To cope with the complexity of human datasets featured by noisy data, nonlinearity and multidimensionality, we assessed various machine-learning methods. Of these, the linear discriminant analysis, Naïve Bayesian model and stochastic gradient boost model yielded perfect or near-perfect sensibility and specificity and revealed that expression levels of the neuroimmune guidance cues SEMA6B, SEMA6D and EPHA2 in circulating monocytes were of predictive values for cardiovascular disease outcome.
Previously, we identified plasma microRNA (miR) profiles that associate with markers of microvascular injury in patients with diabetic nephropathy (DN). However, miRs circulate in extracellular ...vesicles (EVs) or in association with HDL or the RNA-binding protein argonaute-2 (Ago-2). Given that the EV- and HDL-mediated miR transfer toward endothelial cells (ECs) regulates cellular quiescence and inflammation, we hypothesized that the distribution of miRs among carriers affects microvascular homeostasis in DN. Therefore, we determined the miR expression in EV, HDL, and Ago-2 fractions isolated from EDTA plasma of healthy control subjects, patients with diabetes mellitus (DM) with or without early DN (estimated glomerular filtration rate eGFR >30 mL/min/1.73 m
), and patients with DN (eGFR <30 mL/min/1.73 m
). Consistent with our hypothesis, we observed alterations in miR carrier distribution in plasma of patients with DM and DN compared with healthy control subjects. Both miR-21 and miR-126 increased in EVs of patients with DN, whereas miR-660 increased in the Ago-2 fraction and miR-132 decreased in the HDL fraction. Moreover, in vitro, differentially expressed miRs improved EC barrier formation (EV-miR-21) and rescued the angiogenic potential (HDL-miR-132) of ECs cultured in serum from patients with DM and DN. In conclusion, miR measurement in EVs, HDL, and Ago-2 may improve the biomarker sensitivity of these miRs for microvascular injury in DN, while carrier-specific miRs can improve endothelial barrier formation (EV-miR-21/126) or exert a proangiogenic response (HDL-miR-132).
MicroRNAs are negative regulators of gene expression that play a key role in cell‐type specific differentiation and modulation of cell function and have been proposed to be involved in ...neovascularization. Previously, using an extensive cloning and sequencing approach, we identified miR‐126 to be specifically and highly expressed in human endothelial cells (EC). Here, we demonstrate EC‐specific expression of miR‐126 in capillaries and the larger vessels in vivo. We therefore explored the potential role of miR‐126 in arteriogenesis and angiogenesis. Using miR‐reporter constructs, we show that miR‐126 is functionally active in EC in vitro and that it could be specifically repressed using antagomirs specifically targeting miR‐126. To study the consequences of miR‐126 silencing on vascular regeneration, mice were injected with a single dose of antagomir‐126 or a control ‘scramblemir’ and exposed to ischemia of the left hindlimb by ligation of the femoral artery. Although miR‐126 was effectively silenced in mice treated with a single, high dose (HD) of antagomir‐126, laser Doppler perfusion imaging did not show effects on blood flow recovery. In contrast, quantification of the capillary density in the gastrocnemius muscle revealed that mice treated with a HD of antagomir‐126 had a markedly reduced angiogenic response. Aortic explant cultures of the mice confirmed the role of miR‐126 in angiogenesis. Our data demonstrate a facilitary function for miR‐126 in ischemia‐induced angiogenesis and show the efficacy and specificity of antagomir‐induced silencing of EC‐specific microRNAs in vivo.
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.
Chronic kidney disease is associated with progressive renal fibrosis, where perivascular cells give rise to the majority of α-smooth muscle actin (α-SMA) positive myofibroblasts. Here we sought to ...identify pericytic miRNAs that could serve as a target to decrease myofibroblast formation. Kidney fibrosis was induced in FoxD1-GC;Z/Red-mice by unilateral ureteral obstruction followed by FACS sorting of dsRed-positive FoxD1-derivative cells and miRNA profiling. MiR-132 selectively increased 21-fold during pericyte-to-myofibroblast formation, whereas miR-132 was only 2.5-fold up in total kidney lysates (both in obstructive and ischemia-reperfusion injury). MiR-132 silencing during obstruction decreased collagen deposition (35%) and tubular apoptosis. Immunohistochemistry, Western blot, and qRT-PCR confirmed a similar decrease in interstitial α-SMA+ cells. Pathway analysis identified a rate-limiting role for miR-132 in myofibroblast proliferation that was confirmed in vitro. Indeed, antagomir-132–treated mice displayed a reduction in the number of proliferating Ki67+ interstitial myofibroblasts. Interestingly, this was selective for the interstitial compartment and did not impair the reparative proliferation of tubular epithelial cells, as evidenced by an increase in Ki67+ epithelial cells, as well as increased phospho-RB1, Cyclin-A and decreased RASA1, p21 levels in kidney lysates. Additional pathway and gene expression analyses suggest miR-132 coordinately regulates genes involved in TGF-β signaling (Smad2/Smad3), STAT3/ERK pathways, and cell proliferation (Foxo3/p300). Thus, silencing miR-132 counteracts the progression of renal fibrosis by selectively decreasing myofibroblast proliferation and could potentially serve as a novel antifibrotic therapy.
Simultaneous pancreas–kidney transplantation (SPKT) replaces kidney function and restores endogenous insulin secretion in patients with diabetic nephropathy (DN). Here, we aimed to identify ...circulating long noncoding RNAs (lncRNAs) that are associated with DN and vascular injury in the context of SPKT. Based on a pilot study and a literature‐based selection of vascular injury–related lncRNAs, we assessed 9 candidate lncRNAs in plasma samples of patients with diabetes mellitus with a kidney function >35 mL/min/1.73 m2 (DM; n = 12), DN (n = 14), SPKT (n = 35), healthy controls (n = 15), and renal transplant recipients (KTx; n = 13). DN patients were also studied longitudinally before and 1, 6, and 12 months after SPKT. Of 9 selected lncRNAs, we found MALAT1, LIPCAR, and LNC‐EPHA6 to be higher in DN compared with healthy controls. SPKT caused MALAT1, LIPCAR, and LNC‐EPHA6 to normalize to levels of healthy controls, which was confirmed in the longitudinal study. In addition, we observed a strong association between MALAT1, LNC‐EPHA6, and LIPCAR and vascular injury marker soluble thrombomodulin and a subset of angiogenic microRNAs (miR‐27a, miR‐130b, miR‐152, and miR‐340). We conclude that specific circulating lncRNAs associate with DN‐related vascular injury and normalize after SPKT, suggesting that lncRNAs may provide a promising novel monitoring strategy for vascular integrity in the context of SPKT.
Specific long noncoding RNAs elevated in type I diabetic patients with diabetic nephropathy compared to healthy controls and simultaneous kidney–pancreas transplant recipients correlate with vascular injury markers and normalize after kidney–pancreas transplantation but not kidney transplantation alone.
There is an increasing prevalence of chronic kidney disease (CKD), which associates with the development of interstitial fibrosis. Pericytes (perivascular fibroblasts) provide a major source of ...α-SMA-positive myofibroblasts that are responsible for the excessive deposition of extracellular matrix. In order to identify pericyte long non-coding RNAs (lncRNAs) that could serve as a target to decrease myofibroblast formation and counteract the progression of kidney fibrosis we employed two models of experimental kidney injury, one focused on kidney fibrosis (unilateral ureteral obstruction; UUO), and one focused on acute kidney injury that yields kidney fibrosis in the longer term (unilateral ischemia-reperfusion injury; IRI). This was performed in FoxD1-GC;tdTomato stromal cell reporter mice that allowed pericyte fate tracing. Tomato red-positive FoxD1-derivative cells of control and injured kidneys were FACS-sorted and used for lncRNA and mRNA profiling yielding a distinctive transcriptional signature of pericytes and myofibroblasts with 244 and 586 differentially expressed lncRNAs (>twofold,
< 0.05), in the UUO and IRI models, respectively. Next, we selected two differentially expressed and conserved lncRNAs, Rian (RNA imprinted and accumulated in nucleus) and Miat (Myocardial infarction associated transcript), and explored their potential regulatory role in myofibroblast formation through knockdown of their function with gapmers. While Miat was upregulated in myofibroblasts of UUO and IRI in mice, gapmer silencing of Miat attenuated myofibroblast formation as evidenced by decreased expression of
,
,
, and
, as well as decreased α-SMA and pro-collagen-1α1 protein levels. In contrast, silencing Rian, which was found to be downregulated in kidney myofibroblast after IRI and UUO, resulted in increased myofibroblast formation. In addition, we found microRNAs that were previously linked to Miat (miR-150) and Rian (14q32 miRNA cluster), to be dysregulated in the FoxD1-derivative cells, suggesting a possible interaction between miRNAs and these lncRNAs in myofibroblast formation. Taken together, lncRNAs play a regulatory role in myofibroblast formation, possibly through interacting with miRNA regulation, implicating that understanding their biology and their modulation may have the potential to counteract the development of renal fibrosis.
Acute rejection (AR) of a kidney graft in renal transplant recipients is associated with microvascular injury in graft dysfunction and, ultimately, graft failure. Circulating long noncoding RNAs ...(lncRNAs) may be suitable markers for vascular injury in the context of AR. Here, we first investigated the effect of AR after kidney transplantation on local vascular integrity and demonstrated that the capillary density markedly decreased in AR kidney biopsies compared to pre-transplant biopsies. Subsequently, we assessed the circulating levels of four lncRNAs (LNC-RPS24, LNC-EPHA6, MALAT1, and LIPCAR), that were previously demonstrated to associate with vascular injury in a cohort of kidney recipients with a stable kidney transplant function (
= 32) and recipients with AR (
= 15). The latter were followed longitudinally six and 12 months after rejection. We found higher levels of circulating LNC-EPHA6 during rejection, compared with renal recipients with a stable kidney function (
= 0.017), that normalized one year after AR. In addition, LNC-RPS24, LNC-EPHA6, and LIPCAR levels correlated significantly with the vascular injury marker soluble thrombomodulin. We conclude that AR and microvascular injury are associated with higher levels of circulating LNC-EPHA6, which emphasizes the potential role of lncRNAs as biomarker in the context of AR.
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