FAM222A, Part of the BET-Regulated Basal Endothelial Transcriptome, Is a Novel Determinant of Endothelial Biology and Angiogenesis-Brief Report.

BACKGROUND: BETs (bromodomain and extraterminal domain-containing epigenetic reader proteins), including BRD4 (bromodomain-containing protein 4), orchestrate transcriptional programs induced by pathogenic stimuli, as intensively studied in cardiovascular disease and elsewhere. In endothelial cells (ECs), BRD4 directs induced proinflammatory, proatherosclerotic transcriptional responses; BET inhibitors, like JQ1, repress these effects and decrease atherosclerosis. While BET effects in pathogenic conditions have prompted therapeutic BET inhibitor development, BET action under basal conditions, including ECs, has remained understudied. To understand BET action in basal endothelial transcriptional programs, we first analyzed EC RNA-Seq data in the absence versus presence of JQ1 before using BET regulation to identify novel determinants of EC biology and function. METHODS: RNA-Seq datasets of human umbilical vein ECs without and with JQ1 treatment were analyzed. After identifying C12orf34, also known as FAM222A (family with sequence similarity 222 member A), as a previously unreported, basally expressed, potently JQ1-induced EC gene, FAM222A was studied in endothelial and angiogenic responses in vitro using small-interference RNA silencing and lentiviral overexpression, in vitro, ex vivo and in vivo, including aortic sprouting, matrigel plug assays, and murine neonatal oxygen-induced retinopathy. RESULTS: Resting EC RNA-Seq data indicate BETs direct transcriptional programs underlying core endothelial properties including migration, proliferation, and angiogenesis. BET inhibition in resting ECs also significantly induced a subset of mRNAs, including FAM222A-a unique BRD4-regulated gene with no reported EC role. Silencing endothelial FAM222A significantly decreased cellular proliferation, migration, network formation, aorta sprouting, and Matrigel plug vascularization through coordinated modulation of VEGF (vascular endothelial growth factor) and NOTCH mediator expression in vitro, ex vivo, in vivo; lentiviral FAM222A overexpression had opposite effects. In vivo, siFAM222A significantly repressed retinal revascularization in neonatal murine oxygen-induced retinopathy through similar angiogenic signaling modulation. CONCLUSIONS: BET control over the basal endothelial transcriptome includes FAM222A, a novel, BRD4-regulated, key determinant of endothelial biology and angiogenesis.

miR-181b regulates vascular endothelial aging by modulating an MAP3K3 signaling pathway.

Endothelial cell (EC) aging plays a vital role in the pathogenesis of cardiovascular disease (CVD). MicroRNAs have emerged as crucial regulators of target gene expression by inhibiting mRNA translation and/or promoting mRNA degradation. We identify an aging-related and oxidative stress-responsive microRNA, miR-181b, that inhibits endothelial cell apoptosis and senescence. In gain- or loss-of-function studies, miR-181b regulated the expression of key apoptosis markers (Bcl2, Bax, cleaved-Caspase3) and senescence markers (p16, p21, γH2AX) and the ratio of apoptotic cells (TUNEL-positive) and senescent cells (SA-ßgal-positive) in H2 O2 -induced ECs. Mechanistically, miR-181b targets MAP3K3 and modulates a MAP3K3/MKK/MAPK signaling pathway. MAP3K3 knockdown recapitulated the phenotype of miR-181b overexpression and miR-181b was dependent on MAP3K3 for regulating EC apoptosis and senescence. In vivo, miR-181b expression showed a negative correlation with increasing age in the mouse aorta. Endothelial-specific deficiency of miR-181a2b2 increased the target MAP3K3, markers of vascular senescence (p16, p21), and DNA double-strand breaks (γH2AX) in the aorta of aged mice. Collectively, this study unveils an important role of miR-181b in regulating vascular endothelial aging via an MAP3K3-MAPK signaling pathway, providing new potential therapeutic targets for antiaging therapy in CVD.

LncRNA-MAP3K4 regulates vascular inflammation through the p38 MAPK signaling pathway and cis-modulation of MAP3K4.

Chronic vascular inflammation plays a key role in the pathogenesis of atherosclerosis. Long non-coding RNAs (lncRNAs) have emerged as essential inflammation regulators. We identify a novel lncRNA termed lncRNA-MAP3K4 that is enriched in the vessel wall and regulates vascular inflammation. In the aortic intima, lncRNA-MAP3K4 expression was reduced by 50% during the progression of atherosclerosis (chronic inflammation) and 70% during endotoxemia (acute inflammation). lncRNA-MAP3K4 knockdown reduced the expression of key inflammatory factors (eg, ICAM-1, E-selectin, MCP-1) in endothelial cells or vascular smooth muscle cells and decreased monocytes adhesion to endothelium, as well as reducing TNF-α, IL-1ß, COX2 expression in macrophages. Mechanistically, lncRNA-MAP3K4 regulates inflammation through the p38 MAPK signaling pathway. lncRNA-MAP3K4 shares a bidirectional promoter with MAP3K4, an upstream regulator of the MAPK signaling pathway, and regulates its transcription in cis. lncRNA-MAP3K4 and MAP3K4 show coordinated expression in response to inflammation in vivo and in vitro. Similar to lncRNA-MAP3K4, MAP3K4 knockdown reduced the expression of inflammatory factors in several different vascular cells. Furthermore, lncRNA-MAP3K4 and MAP3K4 knockdown showed cooperativity in reducing inflammation in endothelial cells. Collectively, these findings unveil the role of a novel lncRNA in vascular inflammation by cis-regulating MAP3K4 via a p38 MAPK pathway.

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].

LncRNA Meg3 protects endothelial function by regulating the DNA damage response.

The role of long non-coding RNAs (lncRNAs) in regulating endothelial function through the DNA damage response (DDR) remains poorly understood. In this study, we demonstrate that lncRNA maternally expressed gene 3 (Meg3) interacts with the RNA binding protein polypyrimidine tract binding protein 3 (PTBP3) to regulate gene expression and endothelial function through p53 signaling ─ a major coordinator of apoptosis and cell proliferation triggered by the DDR. Meg3 expression is induced in endothelial cells (ECs) upon p53 activation. Meg3 silencing induces DNA damage, activates p53 signaling, increases the expression of p53 target genes, promotes EC apoptosis, and inhibits EC proliferation. Mechanistically, Meg3 silencing reduces the interaction of p53 with Mdm2, induces p53 expression, and promotes the association of p53 with the promoters of a subset of p53 target genes. PTBP3 silencing recapitulates the effects of Meg3 deficiency on the expression of p53 target genes, EC apoptosis and proliferation. The Meg3-dependent association of PTBP3 with the promoters of p53 target genes suggests that Meg3 and PTBP3 restrain p53 activation. Our studies reveal a novel role of Meg3 and PTBP3 in regulating p53 signaling and endothelial function, which may serve as novel targets for therapies to restore endothelial homeostasis.

MiR-4674 regulates angiogenesis in tissue injury by targeting p38K signaling in endothelial cells.

Neoangiogenesis is critical for tissue repair in response to injury such as myocardial ischemia or dermal wound healing. MicroRNAs are small noncoding RNAs and important regulators of angiogenesis under physiological and pathological disease states. Therefore, identification of microRNAs that may restore impaired angiogenesis in response to tissue injury may provide new targets for therapy. Using a microRNA microarray profiling approach, we identified a human-specific microRNA, miR-4674, that was significantly decreased in patients after myocardial tissue injury and had an endothelial cell (EC)-enriched expression pattern. Functionally, overexpression of miR-4674 markedly attenuated EC proliferation, migration, network tube formation, and spheroid sprouting, whereas blockade of miR-4674 had the opposite effects. Transcriptomic profiling, gene set enrichment analyses, bioinformatics, 3'-untranslated region (3'-UTR) reporter and microribonucleoprotein immunoprecipitation (miRNP-IP) assays, and small interfering RNA dependency studies revealed that miR-4674 regulates VEGF stimulated-p38 mitogen-activated protein kinase (MAPK) signaling and targets interleukin 1 receptor-associated kinase 1 (Irak1) and BICD cargo adaptor 2 (Bicd2) in ECs. Furthermore, Irak1 and Bicd2 were necessary for miR-4674-driven EC proliferation and migration. Finally, neutralization of miR-4674 increased angiogenesis, Irak1 and Bicd2 expression, and p38 phosphorylation in human skin organoids as a model of tissue injury. Collectively, targeting miR-4674 may provide a novel therapeutic target for tissue repair in pathological disease states associated with impaired angiogenesis.

MicroRNA-135a-3p regulates angiogenesis and tissue repair by targeting p38 signaling in endothelial cells.

Angiogenesis is a critical process in repair of tissue injury that is regulated by a delicate balance between pro- and antiangiogenic factors. In disease states associated with impaired angiogenesis, we identified that miR-135a-3p is rapidly induced and serves as an antiangiogenic microRNA (miRNA) by targeting endothelial cell (EC) p38 signaling in vitro and in vivo. MiR-135a-3p overexpression significantly inhibited EC proliferation, migration, and network tube formation in matrigel, whereas miR-135-3p neutralization had the opposite effects. Mechanistic studies using transcriptomic profiling, bioinformatics, 3'-UTR reporter and miRNA ribonucleoprotein complex -immunoprecipitation assays, and small interfering RNA dependency studies revealed that miR-135a-3p inhibits the p38 signaling pathway in ECs by targeting huntingtin-interacting protein 1 (HIP1). Local delivery of miR-135a-3p inhibitors to wounds of diabetic db/db mice markedly increased angiogenesis, granulation tissue thickness, and wound closure rates, whereas local delivery of miR-135a-3p mimics impaired these effects. Finally, through gain- and loss-of-function studies in human skin organoids as a model of tissue injury, we demonstrated that miR-135a-3p potently modulated p38 signaling and angiogenesis in response to VEGF stimulation by targeting HIP1. These findings establish miR-135a-3p as a pivotal regulator of pathophysiological angiogenesis and tissue repair by targeting a VEGF-HIP1-p38K signaling axis, providing new targets for angiogenic therapy to promote tissue repair.-Icli, B., Wu, W., Ozdemir, D., Li, H., Haemmig, S., Liu, X., Giatsidis, G., Cheng, H. S., Avci, S. N., Kurt, M., Lee, N., Guimaraes, R. B., Manica, A., Marchini, J. F., Rynning, S. E., Risnes, I., Hollan, I., Croce, K., Orgill, D. P., Feinberg, M. W. MicroRNA-135a-3p regulates angiogenesis and tissue repair by targeting p38 signaling in endothelial cells.

MicroRNA-615-5p Regulates Angiogenesis and Tissue Repair by Targeting AKT/eNOS (Protein Kinase B/Endothelial Nitric Oxide Synthase) Signaling in Endothelial Cells.

Objective- In response to tissue injury, the appropriate progression of events in angiogenesis is controlled by a careful balance between pro and antiangiogenic factors. We aimed to identify and characterize microRNAs that regulate angiogenesis in response to tissue injury. Approach and Results- We show that in response to tissue injury, microRNA-615-5p (miR-615-5p) is rapidly induced and serves as an antiangiogenic microRNA by targeting endothelial cell VEGF (vascular endothelial growth factor)-AKT (protein kinase B)/eNOS (endothelial nitric oxide synthase) signaling in vitro and in vivo. MiR-615-5p expression is increased in wounds of diabetic db/db mice, in plasma of human subjects with acute coronary syndromes, and in plasma and skin of human subjects with diabetes mellitus. Ectopic expression of miR-615-5p markedly inhibited endothelial cell proliferation, migration, network tube formation in Matrigel, and the release of nitric oxide, whereas miR-615-5p neutralization had the opposite effects. Mechanistic studies using transcriptomic profiling, bioinformatics, 3' untranslated region reporter and microribonucleoprotein immunoprecipitation assays, and small interfering RNA dependency studies demonstrate that miR-615-5p inhibits the VEGF-AKT/eNOS signaling pathway in endothelial cells by targeting IGF2 (insulin-like growth factor 2) and RASSF2 (Ras-associating domain family member 2). Local delivery of miR-615-5p inhibitors, markedly increased angiogenesis, granulation tissue thickness, and wound closure rates in db/db mice, whereas miR-615-5p mimics impaired these effects. Systemic miR-615-5p neutralization improved skeletal muscle perfusion and angiogenesis after hindlimb ischemia in db/db mice. Finally, modulation of miR-615-5p expression dynamically regulated VEGF-induced AKT signaling and angiogenesis in human skin organoids as a model of tissue injury. Conclusions- These findings establish miR-615-5p as an inhibitor of VEGF-AKT/eNOS-mediated endothelial cell angiogenic responses and that manipulating miR-615-5p expression could provide a new target for angiogenic therapy in response to tissue injury. Visual Overview- An online visual overview is available for this article.

MicroRNA dysregulation in the tumor microenvironment influences the phenotype of pancreatic cancer.

Cellular interactions in the tumor microenvironment influence neoplastic progression in pancreatic ductal adenocarcinoma. One underlying mechanism is the induction of the prognostically unfavorable epithelial-mesenchymal-transition-like tumor budding. Our aim is to explore the expression of microRNAs implicated in the regulation of tumor budding focusing on the microenvironment of the invasive front. To this end, RNA from laser-capture-microdissected material of the main tumor, tumor buds, juxta-tumoral stroma, tumor-remote stroma, and non-neoplastic pancreatic parenchyma from pancreatic cancer cases with (n=7) and without (n=6) tumor budding was analyzed by qRT-PCR for the expression of a panel of miRNAs that are known to be implicated in the regulation of epithelial-mesenchymal transition, including miR-21, miR-183, miR-200b, miR-200c, miR-203, miR-205, miR-210, and miR-217. Here we show that at the invasive front of pancreatic ductal adenocarcinoma, specific microRNAs, are differentially expressed between tumor buds and main tumor cells and between cases with and without tumor budding, indicating their involvement in the regulation of the budding phenotype. Notably, miR-200b and miR-200c were significantly downregulated in the tumor buds. Consistent with this finding, they negatively correlated with the expression of epithelial-mesenchymal-transition-associated E-cadherin repressors ZEB1 and ZEB2 in the budding cells (P<0.001). Interestingly, many microRNAs were also dysregulated in juxta-tumoral compared to tumor-remote stroma suggesting that juxta-tumoral stroma contributes to microRNA dysregulation. Notably, miR-200b and miR-200c were strongly downregulated while miR-210 and miR-21 were upregulated in the juxta-tumoral vs tumor-remote stroma in carcinomas with tumor budding. In conclusion, microRNA targeting in both tumor and stromal cells could represent a treatment option for aggressive pancreatic cancer.

Long noncoding RNAs in cardiovascular disease, diagnosis, and therapy.

PURPOSE OF REVIEW: Long noncoding RNAs (lncRNAs) have emerged as powerful regulators of nearly all biological processes. Their cell-type and tissue-specific expression in health and disease provides new avenues for diagnosis and therapy. This review highlights the role of lncRNAs that are involved in cardiovascular disease (CVD) with a special focus on cell types involved in cardiac injury and remodeling, vascular injury, angiogenesis, inflammation, and lipid metabolism. RECENT FINDINGS: Almost 98% of the genome does not encode for proteins. LncRNAs are among the most abundant type of RNA in the noncoding genome. Accumulating studies have uncovered novel lncRNA-mediated regulation of CVD-associated genes, signaling pathways, and pathophysiological responses. Targeting lncRNAs in vivo using short antisense oligonucleotides or by gene editing has provided important insights into disease pathogenesis through epigenetic, transcriptional, or translational mechanisms. Although cross-species conservation still remains a major obstacle, there is increasing appreciation that altered expression of lncRNAs associates with stage-specific CVD and in human patient cohorts, providing new opportunities for diagnosis and therapy. SUMMARY: A better understanding of lncRNAs will not only fundamentally improve our understanding of key signaling pathways in CVD, but also aid in the development of effective new therapies and RNA-based biomarkers.

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.

Endothelial cell-specific deletion of a microRNA accelerates atherosclerosis.

BACKGROUND AND AIMS: Chronic vascular endothelial inflammation predisposes to atherosclerosis; however, the cell-autonomous roles for endothelial-expressing microRNAs (miRNAs) are poorly understood in this process. MiR-181b is expressed in several cellular constituents relevant to lesion formation. The aim of this study is to examine the role of genetic deficiency of the miR-181b locus in endothelial cells during atherogenesis. METHODS AND RESULTS: Using a proprotein convertase subtilisin/kexin type 9 (PCSK9)-induced atherosclerosis mouse model, we demonstrated that endothelial cell (EC)-specific deletion of miR-181a2b2 significantly promoted atherosclerotic lesion formation, cell adhesion molecule expression, and the influx of lesional macrophages in the vessel wall. Yet, endothelium deletion of miR-181a2b2 did not affect body weight, lipid metabolism, anti-inflammatory Ly6Clow or the pro-inflammatory Ly6Cinterm and Ly6Chigh fractions in circulating peripheral blood mononuclear cells (PBMCs), and pro-inflammatory or anti-inflammatory mediators in both bone marrow (BM) and PBMCs. Mechanistically, bulk RNA-seq and gene set enrichment analysis of ECs enriched from the aortic arch intima, as well as single cell RNA-seq from atherosclerotic lesions, revealed that endothelial miR-181a2b2 serves as a critical regulatory hub in controlling endothelial inflammation, cell adhesion, cell cycle, and immune response during atherosclerosis. CONCLUSIONS: Our study establishes that deficiency of a miRNA specifically in the vascular endothelium is sufficient to profoundly impact atherogenesis. Endothelial miR-181a2b2 deficiency regulates multiple key pathways related to endothelial inflammation, cell adhesion, cell cycle, and immune response involved in the development of atherosclerosis.

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.

Methotrexate attenuates vascular inflammation through an adenosine-microRNA-dependent pathway.

Endothelial cell (EC) activation is an early hallmark in the pathogenesis of chronic vascular diseases. MicroRNA-181b (Mir181b) is an important anti-inflammatory mediator in the vascular endothelium affecting endotoxemia, atherosclerosis, and insulin resistance. Herein, we identify that the drug methotrexate (MTX) and its downstream metabolite adenosine exert anti-inflammatory effects in the vascular endothelium by targeting and activating Mir181b expression. Both systemic and endothelial-specific Mir181a2b2-deficient mice develop vascular inflammation, white adipose tissue (WAT) inflammation, and insulin resistance in a diet-induced obesity model. Moreover, MTX attenuated diet-induced WAT inflammation, insulin resistance, and EC activation in a Mir181a2b2-dependent manner. Mechanistically, MTX attenuated cytokine-induced EC activation through a unique adenosine-adenosine receptor A3-SMAD3/4-Mir181b signaling cascade. These findings establish an essential role of endothelial Mir181b in controlling vascular inflammation and that restoring Mir181b in ECs by high-dose MTX or adenosine signaling may provide a potential therapeutic opportunity for anti-inflammatory therapy.

LncRNA VINAS regulates atherosclerosis by modulating NF-κB and MAPK signaling.

Long noncoding RNAs (lncRNAs) play important roles in regulating diverse cellular processes in the vessel wall, including atherosclerosis. RNA-Seq profiling of intimal lesions revealed a lncRNA, VINAS (Vascular INflammation and Atherosclerosis lncRNA Sequence), that is enriched in the aortic intima and regulates vascular inflammation. Aortic intimal expression of VINAS fell with atherosclerotic progression and rose with regression. VINAS knockdown reduced atherosclerotic lesion formation by 55% in LDL receptor-deficient (LDLR-/-) mice, independent of effects on circulating lipids, by decreasing inflammation in the vessel wall. Loss- and gain-of-function studies in vitro demonstrated that VINAS serves as a critical regulator of inflammation by modulating NF-κB and MAPK signaling pathways. VINAS knockdown decreased the expression of key inflammatory markers, such as MCP-1, TNF-α, IL-1ß, and COX-2, in endothelial cells (ECs), vascular smooth muscle cells, and bone marrow-derived macrophages. Moreover, VINAS silencing decreased expression of leukocyte adhesion molecules VCAM-1, E-selectin, and ICAM-1 and reduced monocyte adhesion to ECs. DEP domain containing 4 (DEPDC4), an evolutionary conserved human ortholog of VINAS with approximately 74% homology, showed similar regulation in human and pig atherosclerotic specimens. DEPDC4 knockdown replicated antiinflammatory effects of VINAS in human ECs. These findings reveal a potentially novel lncRNA that regulates vascular inflammation, with broad implications for vascular diseases.

A macrophage-specific lncRNA regulates apoptosis and atherosclerosis by tethering HuR in the nucleus.

Long non-coding RNAs (lncRNAs) are emerging regulators of pathophysiological processes including atherosclerosis. Using RNA-seq profiling of the intima of lesions, here we identify a macrophage-specific lncRNA MAARS (Macrophage-Associated Atherosclerosis lncRNA Sequence). Aortic intima expression of MAARS increases by 270-fold with atherosclerotic progression and decreases with regression by 60%. MAARS knockdown reduces atherosclerotic lesion formation by 52% in LDLR-/- mice, largely independent of effects on lipid profile and inflammation, but rather by decreasing macrophage apoptosis and increasing efferocytosis in the vessel wall. MAARS interacts with HuR/ELAVL1, an RNA-binding protein and important regulator of apoptosis. Overexpression and knockdown studies verified MAARS as a critical regulator of macrophage apoptosis and efferocytosis in vitro, in an HuR-dependent manner. Mechanistically, MAARS knockdown alters HuR cytosolic shuttling, regulating HuR targets such as p53, p27, Caspase-9, and BCL2. These findings establish a mechanism by which a macrophage-specific lncRNA interacting with HuR regulates apoptosis, with implications for a broad range of vascular disease states.

KLF10 Deficiency in CD4+ T Cells Triggers Obesity, Insulin Resistance, and Fatty Liver.

CD4+ T cells regulate inflammation and metabolism in obesity. An imbalance of CD4+ T regulatory cells (Tregs) is critical in the development of insulin resistance and diabetes. Although cytokine control of this process is well understood, transcriptional regulation is not. KLF10, a member of the Kruppel-like transcription factor family, is an emerging regulator of immune cell function. We generated CD4+-T-cell-specific KLF10 knockout (TKO) mice and identified a predisposition to obesity, insulin resistance, and fatty liver due to defects of CD4+ Treg mobilization to liver and adipose tissue depots and decreased transforming growth factor ß3 (TGF-ß3) release in vitro and in vivo. Adoptive transfer of wild-type CD4+ Tregs fully rescued obesity, insulin resistance, and fatty liver. Mechanistically, TKO Tregs exhibit reduced mitochondrial respiration and glycolysis, phosphatidylinositol 3-kinase (PI3K)-Akt-mTOR signaling, and consequently impaired chemotactic properties. Collectively, our study identifies CD4+ T cell KLF10 as an essential regulator of obesity and insulin resistance by altering Treg metabolism and mobilization.