A miRNA cassette reprograms smooth muscle cells into endothelial cells.

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.

Deficiency of lncRNA SNHG12 impairs ischemic limb neovascularization by altering an endothelial cell cycle pathway.

SNHG12, a long noncoding RNA (lncRNA) dysregulated in atherosclerosis, is known to be a key regulator of vascular senescence in endothelial cells (ECs). However, its role in angiogenesis and peripheral artery disease has not been elucidated. Hind-limb ischemia studies using femoral artery ligation (FAL) in mice showed that SNHG12 expression falls readily in the acute phase of the response to limb ischemia in gastrocnemius muscle and recovers to normal when blood flow recovery is restored to ischemic muscle, indicating that it likely plays a role in the angiogenic response to ischemia. Gain- and loss-of-function studies demonstrated that SNHG12 regulated angiogenesis - SNHG12 deficiency reduced cell proliferation, migration, and endothelial sprouting, whereas overexpression promoted these angiogenic functions. We identified SNHG12 binding partners by proteomics that may contribute to its role in angiogenesis, including IGF-2 mRNA-binding protein 3 (IGF2BP3, also known as IMP3). RNA-Seq profiling of SNHG12-deficient ECs showed effects on angiogenesis pathways and identified a strong effect on cell cycle regulation, which may be modulated by IMP3. Knockdown of SNHG12 in mice undergoing FAL using injected gapmeRs) decreased angiogenesis, an effect that was more pronounced in a model of insulin-resistant db/db mice. RNA-Seq profiling of the EC and non-EC compartments in these mice revealed a likely role of SNHG12 knockdown on Wnt, Notch, and angiopoietin signaling pathways. Together, these findings indicate that SNHG12 plays an important role in the angiogenic EC response to ischemia.

A Smooth Muscle Cell-Enriched Long Noncoding RNA Regulates Cell Plasticity and Atherosclerosis by Interacting With Serum Response Factor.

Objective: 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. Approach and Results: 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-ß1 (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 600­1197 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. Conclusions: 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.

Novel Lesional Transcriptional Signature Separates Atherosclerosis With and Without Diabetes in Yorkshire Swine and Humans.

[Figure: see text].

Long noncoding RNA SNHG12 integrates a DNA-PK-mediated DNA damage response and vascular senescence.

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 Ldlr -/- mice on a high-cholesterol diet during lesion progression and regression phases. We found that the evolutionarily conserved lncRNA small nucleolar host gene-12 (SNHG12) is highly expressed in the vascular endothelium and decreases during lesion progression. SNHG12 knockdown accelerated atherosclerotic lesion formation by 2.4-fold in Ldlr -/- 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 SNHG12 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 SNHG12 interacted with DNA-dependent protein kinase (DNA-PK), an important regulator of the DNA damage response. The absence of SNHG12 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 SNHG12 knockdown. SNHG12 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.

MicroRNA-181b inhibits thrombin-mediated endothelial activation and arterial thrombosis by targeting caspase recruitment domain family member 10.

Thrombogenic and inflammatory mediators, such as thrombin, induce NF-κB-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-κB signaling in response to TNF-α. Here, we demonstrate that miR-181b uniquely inhibits upstream NF-κB signaling in response to thrombin. Overexpression of miR-181b inhibited thrombin-induced activation of NF-κB signaling, demonstrated by reduction of phospho-IKK-ß, -IκB-α, and p65 nuclear translocation in ECs. MiR-181b also reduced expression of NF-κB 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. 3'-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-κB signaling and targets. Card10 deficiency did not affect TNF-α-induced activation of NF-κB signaling, which suggested stimulus-specific regulation of NF-κB 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.

Regulation of impaired angiogenesis in diabetic dermal wound healing by microRNA-26a.

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.

Systemic delivery of microRNA-181b inhibits nuclear factor-κB activation, vascular inflammation, and atherosclerosis in apolipoprotein E-deficient mice.

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.

MicroRNA-26a regulates pathological and physiological angiogenesis by targeting BMP/SMAD1 signaling.

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(WAF/CIP) 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.

Gene expression in endothelial cells and intimal smooth muscle cells in atherosclerosis-prone or atherosclerosis-resistant regions of the human aorta.

BACKGROUND/AIMS: We compared the atherogenic gene expression in the intimas of atherosclerosis-prone regions (proximal walls), which are exposed to disturbed shear stress, and atherosclerosis-resistant regions (apices), which are exposed to unidirectional laminar shear stress, at the orifices of the intercostal arteries of human aortas. METHODS AND RESULTS: Expression of mRNAs, detected by in situ RT-PCR, for IL-1 beta, TNF-alpha, VCAM-1, PAF receptor and GRP in endothelial cells (ECs), and of PDGF receptor beta (PDGFR-beta), MCP-1, GRP and collagen type-1 by smooth muscle cells (SMCs) in the proximal walls, was significantly enhanced, while seldom observed in the elastic-hyperplastic layer of the apices. Protein expression of PDGFR-beta, IL-1 beta and TNF-alpha was also observed on the proximal walls. SMC growth in the apices was inhibited. Cultured SMC growth and their expression of PDGFR-beta were also significantly inhibited by elastin. CONCLUSION: These results suggest that the construction of the elastic-hyperplastic layer and the subsequent inhibition of SMC growth by elastin, with stabilized ECs under unidirectional laminar shear stress, resulted in atherosclerosis-resistant regions at the apices of human aortas, and that the continuous induction of atherogenic gene expression by ECs activated by disturbed shear stress inhibits the formation of atherosclerosis-resistant intima along the proximal walls.

The integrin-linked kinase regulates cell morphology and motility in a rho-associated kinase-dependent manner.

The integrin-linked kinase (ILK) is a multidomain focal adhesion protein implicated in signal transmission from integrin and growth factor receptors. We have determined that ILK regulates U2OS osteosarcoma cell spreading and motility in a manner requiring both kinase activity and localization. Overexpression of wild-type (WT) ILK resulted in suppression of cell spreading, polarization, and motility to fibronectin. Cell lines overexpressing kinase-dead (S343A) or paxillin binding site mutant ILK proteins display inhibited haptotaxis to fibronectin. Conversely, spreading and motility was potentiated in cells expressing the "dominant negative," non-targeting, kinase-deficient E359K ILK protein. Suppression of cell spreading and motility of WT ILK U2OS cells could be rescued by treatment with the Rho-associated kinase (ROCK) inhibitor Y-27632 or introduction of dominant negative ROCK or RhoA, suggesting these cells have increased RhoA signaling. Activation of focal adhesion kinase (FAK), a negative regulator of RhoA, was reduced in WT ILK cells, whereas overexpression of FAK rescued the observed defects in spreading and cell polarity. Thus, ILK-dependent effects on ROCK and/or RhoA signaling may be mediated through FAK.