RATIONALE:Activated nuclear factor (NF)-κB signaling in the vascular endothelium promotes the initiation and progression of atherosclerosis. Targeting endothelial NF-κB may provide a novel strategy ...to limit chronic inflammation.
OBJECTIVE:To examine the role of microRNA-181b (miR-181b) in endothelial NF-κB signaling and effects on atherosclerosis.
METHODS AND RESULTS:MiR-181b expression was reduced in the aortic intima and plasma in apolipoprotein E–deficient mice fed a high-fat diet. Correspondingly, circulating miR-181b in the plasma was markedly reduced in human subjects with coronary artery disease. Systemic delivery of miR-181b resulted in a 2.3-fold overexpression of miR-181b in the aortic intima of apolipoprotein E–deficient mice and suppressed NF-κB signaling revealed by bioluminescence imaging and reduced target gene expression in the aortic arch in apolipoprotein E–deficient/NF-κB-luciferase transgenic mice. MiR-181b significantly inhibited atherosclerotic lesion formation, proinflammatory gene expression and the influx of lesional macrophages and CD4+ T cells in the vessel wall. Mechanistically, miR-181b inhibited the expression of the target gene importin-α3, an effect that reduced NF-κB nuclear translocation specifically in the vascular endothelium of lesions, whereas surprisingly leukocyte NF-κB signaling was unaffected despite a 7-fold overexpression of miR-181b. Our findings uncover that NF-κB nuclear translocation in leukocytes does not involve importin-α3, but rather importin-α5, which miR-181b does not target, highlighting that inhibition of NF-κB signaling in the endothelium is sufficient to mediate miR-181b’s protective effects.
CONCLUSIONS:Systemic delivery of miR-181b inhibits the activation of NF-κB and atherosclerosis through cell-specific mechanisms in the vascular endothelium. These findings support the rationale that delivery of miR-181b may provide a novel therapeutic approach to treat chronic inflammatory diseases such as atherosclerosis.
Abstract Wound healing is a physiological reparative response to injury and a well-orchestrated process that involves hemostasis, cellular migration, proliferation, angiogenesis, extracellular matrix ...deposition, and wound contraction and re-epithelialization. However, patients with type 2 diabetes mellitus (T2D) are frequently afflicted with impaired wound healing that progresses into chronic wounds or diabetic ulcers, and may lead to complications including limb amputation. Herein, we investigate the potential role of microRNA-26a (miR-26a) in a diabetic model of wound healing. Expression of miR-26a is rapidly induced in response to high glucose in endothelial cells (ECs). Punch skin biopsy wounding of db/db mice revealed increased expression of miR-26a (~ 3.5-fold) four days post-wounding compared to that of WT mice. Local administration of a miR-26a inhibitor, LNA-anti-miR-26a, induced angiogenesis (up to ~ 80%), increased granulation tissue thickness (by 2.5-fold) and accelerated wound closure (53% after nine days) compared to scrambled anti-miR controls in db/db mice. These effects were independent of altered M1/M2 macrophage ratios. Mechanistically, inhibition of miR-26a increased its target gene SMAD1 in ECs nine days post-wounding of diabetic mice. In addition, high glucose reduced activity of the SMAD1-3′-UTR. Diabetic dermal wounds treated with LNA-anti-miR-26a had increased expression of ID1, a downstream modulator or SMAD1, and decreased expression of the cell cycle inhibitor p27. These findings establish miR-26a as an important regulator on the progression of skin wounds of diabetic mice by specifically regulating the angiogenic response after injury, and demonstrate that neutralization of miR-26a may serve as a novel approach for therapy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Long noncoding RNAs (lncRNAs) are emerging regulators of biological processes in the vessel wall; however, their role in atherosclerosis remains poorly defined. We used RNA sequencing to profile ...lncRNAs derived specifically from the aortic intima of
mice on a high-cholesterol diet during lesion progression and regression phases. We found that the evolutionarily conserved lncRNA small nucleolar host gene-12 (
) is highly expressed in the vascular endothelium and decreases during lesion progression.
knockdown accelerated atherosclerotic lesion formation by 2.4-fold in
mice by increased DNA damage and senescence in the vascular endothelium, independent of effects on lipid profile or vessel wall inflammation. Conversely, intravenous delivery of
protected the tunica intima from DNA damage and atherosclerosis. LncRNA pulldown in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that
interacted with DNA-dependent protein kinase (DNA-PK), an important regulator of the DNA damage response. The absence of
reduced the DNA-PK interaction with its binding partners Ku70 and Ku80, abrogating DNA damage repair. Moreover, the anti-DNA damage agent nicotinamide riboside (NR), a clinical-grade small-molecule activator of NAD
, fully rescued the increases in lesional DNA damage, senescence, and atherosclerosis mediated by
knockdown.
expression was also reduced in pig and human atherosclerotic specimens and correlated inversely with DNA damage and senescent markers. These findings reveal a role for this lncRNA in regulating DNA damage repair in the vessel wall and may have implications for chronic vascular disease states and aging.
RATIONALE:The rapid induction and orchestration of new blood vessels are critical for tissue repair in response to injury, such as myocardial infarction, and for physiological angiogenic responses, ...such as embryonic development and exercise.
OBJECTIVE:We aimed to identify and characterize microRNAs (miR) that regulate pathological and physiological angiogenesis.
METHODS AND RESULTS:We show that miR-26a regulates pathological and physiological angiogenesis by targeting endothelial cell (EC) bone morphogenic protein/SMAD1 signaling in vitro and in vivo. MiR-26a expression is increased in a model of acute myocardial infarction in mice and in human subjects with acute coronary syndromes. Ectopic expression of miR-26a markedly induced EC cycle arrest and inhibited EC migration, sprouting angiogenesis, and network tube formation in matrigel, whereas blockade of miR-26a had the opposite effects. Mechanistic studies demonstrate that miR-26a inhibits the bone morphogenic protein/SMAD1 signaling pathway in ECs by binding to the SMAD1 3′-untranslated region, an effect that decreased expression of Id1 and increased p21 and p27. In zebrafish, miR-26a overexpression inhibited formation of the caudal vein plexus, a bone morphogenic protein-responsive process, an effect rescued by ectopic SMAD1 expression. In mice, miR-26a overexpression inhibited EC SMAD1 expression and exercise-induced angiogenesis. Furthermore, systemic intravenous administration of an miR-26a inhibitor, locked nucleic acid-anti–miR-26a, increased SMAD1 expression and rapidly induced robust angiogenesis within 2 days, an effect associated with reduced myocardial infarct size and improved heart function.
CONCLUSIONS:These findings establish miR-26a as a regulator of bone morphogenic protein/SMAD1-mediated EC angiogenic responses, and that manipulating miR-26a expression could provide a new target for rapid angiogenic therapy in ischemic disease states.
Vascular smooth muscle cell (VSMC) plasticity plays a critical role in the development of atherosclerosis. Long noncoding RNAs (lncRNAs) are emerging as important regulators in the vessel wall and ...impact cellular function through diverse interactors. However, the role of lncRNAs in regulating VSMCs plasticity and atherosclerosis remains unclear.
We identified a VSMC-enriched lncRNA cardiac mesoderm enhancer-associated noncoding RNA (CARMN) that is dynamically regulated with progression of atherosclerosis. In both mouse and human atherosclerotic plaques, CARMN colocalized with VSMCs and was expressed in the nucleus. Knockdown of CARMN using antisense oligonucleotides in Ldlr−/− mice significantly reduced atherosclerotic lesion formation by 38% and suppressed VSMCs proliferation by 45% without affecting apoptosis. In vitro CARMN gain- and loss-of-function studies verified effects on VSMC proliferation, migration, and differentiation. TGF-β1 (transforming growth factor-beta) induced CARMN expression in a Smad2/3-dependent manner. CARMN regulated VSMC plasticity independent of the miR143/145 cluster, which is located in close proximity to the CARMN locus. Mechanistically, lncRNA pulldown in combination with mass spectrometry analysis showed that the nuclear-localized CARMN interacted with SRF (serum response factor) through a specific 600–1197 nucleotide domain. CARMN enhanced SRF occupancy on the promoter regions of its downstream VSMC targets. Finally, knockdown of SRF abolished the regulatory role of CARMN in VSMC plasticity.
The lncRNA CARMN is a critical regulator of VSMC plasticity and atherosclerosis. These findings highlight the role of a lncRNA in SRF-dependent signaling and provide implications for a range of chronic vascular occlusive disease states.
ABSTRACT
Thrombogenic and inflammatory mediators, such as thrombin, induce NF‐kB–mediated endothelial cell (EC) activation and dysfunction, which contribute to pathogenesis of arterial thrombosis. ...The role of anti‐inflammatory microRNA‐181b (miR‐181b) on thrombosis remains unknown. Our previous study demonstrated that miR‐181b inhibits downstream NF‐kB signaling in response to TNF‐a. Here, we demonstrate that miR‐181b uniquely inhibits upstream NF‐kB signaling in response to thrombin. Overexpression of miR‐181b inhibited thrombin‐induced activation of NF‐kB signaling, demonstratedbyreductionofphospho‐IKK‐b,‐IkB‐a, and p65 nuclear translocation in ECs. MiR‐181b also reduced expression of NF‐kB target genes VCAM‐1, intercellular adhesion molecule‐1, E‐selectin, and tissue factor. Mechanistically, miR‐181b targets caspase recruitment domain family member 10 (Card10), an adaptor protein that participates in activation of the IKK complex in response to signals transduced from protease‐activated receptor‐1. miR‐181b reduced expression of Card10 mRNA and protein, but not protease‐activated receptor‐1. 39‐Untranslated region reporter assays, argonaute‐2 microribonucleoprotein immunoprecipitation studies, and Card10 rescue studies revealed that Card10 is a bona fide direct miR‐181b target. Small interfering RNA–mediated knockdown of Card10 expression phenocopied effects of miR‐181b on NF‐kB signaling and targets. Card10 deficiency did not affect TNF‐a–induced activation of NF‐kB signaling, which suggested stimulus‐specific regulation of NF‐kB signaling and endothelial responses by miR‐181b in ECs. Finally, in response to photochemical injury‐induced arterial thrombosis, systemic delivery of miR‐181b reduced thrombus formation by 73% in carotid arteries and prolonged time to occlusion by 1.6‐fold, effects recapitulated by Card10 small interfering RNA. These data demonstrate that miR‐181b and Card10 are important regulators of thrombin‐induced EC activation and arterial thrombosis. These studies highlight the relevance of microRNA‐dependent targets in response to ligand‐specific signaling in ECs.—Lin, J., He, S., Sun, X., Franck, G., Deng, Y., Yang, D., Haemmig, S., Wara, A. K. M., Icli, B., Li, D., Feinberg, M. W. MicroRNA‐181b inhibits thrombin‐mediated endothelial activation and arterial thrombosis by targeting caspase recruitment domain family member 10. FASEB J. 30, 3216–3226 (2016). www.fasebj.org
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Cellular reprogramming through targeting microRNAs (miRNAs) holds promise for regenerative therapy due to their profound regulatory effects in proliferation, differentiation, and function. We ...hypothesized that transdifferentiation of vascular smooth muscle cells (SMCs) into endothelial cells (ECs) using a miRNA cassette may provide a novel approach for use in vascular disease states associated with endothelial injury or dysfunction. miRNA profiling of SMCs and ECs and iterative combinatorial miRNA transfections of human coronary SMCs revealed a 4‐miRNA cassette consisting of miR‐143‐3p and miR‐145‐5p inhibitors and miR‐146a‐5p and miR‐181b‐5p mimics that efficiently produced induced endothelial cells (iECs). Transcriptome profiling, protein expression, and functional studies demonstrated that iECs exhibit high similarity to ECs. Injected iECs restored blood flow recovery even faster than conventional ECs in a murine hindlimb ischemia model. This study demonstrates that a 4‐miRNA cassette is sufficient to reprogram SMCs into ECs and shows promise as a novel regenerative strategy for endothelial repair.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
To determine if adjuvant interstitial hyperthermia (HT) significantly improves survival of patients with glioblastoma undergoing brachytherapy boost after conventional radiotherapy.
Adults with ...newly-diagnosed, focal, supratentorial glioblastoma < or = 5 cm in diameter were registered postoperatively on a Phase II/III randomized trial and treated with partial brain radiotherapy to 59.4 Gy with oral hydroxyurea. Those patients whose tumor was still implantable after teletherapy were randomized to brachytherapy boost (60 Gy at 0.40-0.60 Gy/h) +/- HT for 30 min immediately before and after brachytherapy. Time to progression (TTP) and survival from date of diagnosis were estimated using the Kaplan-Meier method.
From 1990 to 1995, 112 eligible patients were entered in the trial. Patient ages ranged from 21-78 years (median, 54 years) and KPS ranged from 70-100 (median, 90). Most commonly due to tumor progression or patient refusal, 33 patients were never randomized. Of the patients, 39 were randomized to brachytherapy ("no heat") and 40 to brachytherapy + HT ("heat"). By intent to treat, TTP and survival were significantly longer for "heat" than "no heat" (p = 0.04 and p = 0.04). For the 33 "no heat" patients and 35 "heat" patients who underwent brachytherapy boost, TTP and survival were significantly longer for "heat" than "no heat" (p = 0.045 and p = 0.02, respectively; median survival 85 weeks vs. 76 weeks; 2-year survival 31% vs. 15%). A multivariate analysis for these 68 patients adjusting for age and KPS showed that improved survival was significantly associated with randomization to "heat" (p = 0.008; hazard ratio 0.51). There were no Grade 5 toxicities, 2 Grade 4 toxicities (1 on each arm), and 7 Grade 3 toxicities (1 on "no heat" and 6 on the "heat" arm).
Adjuvant interstitial brain HT, given before and after brachytherapy boost, after conventional radiotherapy significantly improves survival of patients with focal glioblastoma, with acceptable toxicity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Activation of macrophages is important in chronic inflammatory disease states such as atherosclerosis. Proinflammatory cytokines such as interferon-γ (IFN-γ), lipopolysaccharide (LPS), or tumor ...necrosis factor-α can promote macrophage activation. Conversely, anti-inflammatory factors such as transforming growth factor-β1 (TGF-β1) can decrease proinflammatory activation. The molecular mediators regulating the balance of these opposing effectors remain incompletely understood. Herein, we identify Kruppel-like factor 4 (KLF4) as being markedly induced in response to IFN-γ, LPS, or tumor necrosis factor-α and decreased by TGF-β1 in macrophages. Overexpression of KLF4 in J774a macrophages induced the macrophage activation marker inducible nitric-oxide synthase and inhibited the TGF-β1 and Smad3 target gene plasminogen activator inhibitor-1 (PAI-1). Conversely, KLF4 knockdown markedly attenuated the ability of IFN-γ, LPS, or IFN-γ plus LPS to induce the iNOS promoter, whereas it augmented macrophage responsiveness to TGF-β1 and Smad3 signaling. The KLF4 induction of the iNOS promoter is mediated by two KLF DNA-binding sites at –95 and –212 bp, and mutation of these sites diminished induction by IFN-γ and LPS. We further provide evidence that KLF4 interacts with the NF-κB family member p65 (RelA) to cooperatively induce the iNOS promoter. In contrast, KLF4 inhibited the TGF-β1/Smad3 induction of the PAI-1 promoter independent of KLF4 DNA binding through a novel antagonistic competition with Smad3 for the C terminus of the coactivator p300/CBP. These findings support an important role for KLF4 as a regulator of key signaling pathways that control macrophage activation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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