A novel mouse model carrying a gene trap insertion into the Hmgxb4 gene locus to examine Hmgxb4 expression in vivo.

HMG (high mobility group) proteins are a diverse family of nonhistone chromosomal proteins that interact with DNA and a wide range of transcriptional regulators to regulate the structural architecture of DNA. HMGXB4 (also known as HMG2L1) is an HMG protein family member that contains a single HMG box domain. Our previous studies have demonstrated that HMGXB4 suppresses smooth muscle differentiation and exacerbates endotoxemia by promoting a systemic inflammatory response in mice. However, the expression of Hmgxb4 in vivo has not fully examined. Herein, we generated a mouse model that harbors a gene trap in the form of a lacZ gene insertion into the Hmgxb4 gene. This mouse enables the visualization of endogenous HMGXB4 expression in different tissues via staining for the ß-galactosidase activity of LacZ which is under the control of the endogenous Hmgxb4 gene promoter. We found that HMGXB4 is widely expressed in mouse tissues and is a nuclear protein. Furthermore, the Hmgxb4 gene trap mice exhibit normal cardiac function and blood pressure. Measurement of ß-galactosidase activity in the Hmgxb4 gene trap mice demonstrated that the arterial injury significantly induces Hmgxb4 expression. In summary, the Hmgxb4 gene trap reporter mouse described here provides a valuable tool to examine the expression level of endogenous Hmgxb4 in both physiological and pathological settings in vivo.

PDZ-Binding Kinase, a Novel Regulator of Vascular Remodeling in Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is high blood pressure in the lungs that originates from structural changes in small resistance arteries. A defining feature of PAH is the inappropriate remodeling of pulmonary arteries (PA) leading to right ventricle failure and death. Although treatment of PAH has improved, the long-term prognosis for patients remains poor, and more effective targets are needed. METHODS: Gene expression was analyzed by microarray, RNA sequencing, quantitative polymerase chain reaction, Western blotting, and immunostaining of lung and isolated PA in multiple mouse and rat models of pulmonary hypertension (PH) and human PAH. PH was assessed by digital ultrasound, hemodynamic measurements, and morphometry. RESULTS: Microarray analysis of the transcriptome of hypertensive rat PA identified a novel candidate, PBK (PDZ-binding kinase), that was upregulated in multiple models and species including humans. PBK is a serine/threonine kinase with important roles in cell proliferation that is minimally expressed in normal tissues but significantly increased in highly proliferative tissues. PBK was robustly upregulated in the medial layer of PA, where it overlaps with markers of smooth muscle cells. Gain-of-function approaches show that active forms of PBK increase PA smooth muscle cell proliferation, whereas silencing PBK, dominant negative PBK, and pharmacological inhibitors of PBK all reduce proliferation. Pharmacological inhibitors of PBK were effective in PH reversal strategies in both mouse and rat models, providing translational significance. In a complementary genetic approach, PBK was knocked out in rats using CRISPR/Cas9 editing, and loss of PBK prevented the development of PH. We found that PBK bound to PRC1 (protein regulator of cytokinesis 1) in PA smooth muscle cells and that multiple genes involved in cytokinesis were upregulated in experimental models of PH and human PAH. Active PBK increased PRC1 phosphorylation and supported cytokinesis in PA smooth muscle cells, whereas silencing or dominant negative PBK reduced cytokinesis and the number of cells in the G2/M phase of the cell cycle. CONCLUSIONS: PBK is a newly described target for PAH that is upregulated in proliferating PA smooth muscle cells, where it contributes to proliferation through changes in cytokinesis and cell cycle dynamics to promote medial thickening, fibrosis, increased PA resistance, elevated right ventricular systolic pressure, right ventricular remodeling, and PH.

The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy.

Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. While the role of ubiquitin (Ub) ligase PARKIN in mitophagy has been extensively studied, increasing evidence suggests the existence of PARKIN-independent mitophagy in highly metabolically active organs such as the heart. Here, we identify a crucial role for Cullin-RING Ub ligase 5 (CRL5) in basal mitochondrial turnover in cardiomyocytes. CRL5 is a multi-subunit Ub ligase comprised by the catalytic RING box protein RBX2 (also known as SAG), scaffold protein Cullin 5 (CUL5), and a substrate-recognizing receptor. Analysis of the mitochondrial outer membrane-interacting proteome uncovered a robust association of CRLs with mitochondria. Subcellular fractionation, immunostaining, and immunogold electron microscopy established that RBX2 and Cul5, two core components of CRL5, localizes to mitochondria. Depletion of RBX2 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial membrane potential and respiration, and increased cell death in cardiomyocytes. In vivo , deletion of the Rbx2 gene in adult mouse hearts suppressed mitophagic activity, provoked accumulation of damaged mitochondria in the myocardium, and disrupted myocardial metabolism, leading to rapid development of dilated cardiomyopathy and heart failure. Similarly, ablation of RBX2 in the developing heart resulted in dilated cardiomyopathy and heart failure. Notably, the action of RBX2 in mitochondria is not dependent on PARKIN, and PARKIN gene deletion had no impact on the onset and progression of cardiomyopathy in RBX2-deficient hearts. Furthermore, RBX2 controls the stability of PINK1 in mitochondria. Proteomics and biochemical analyses further revealed a global impact of RBX2 deficiency on the mitochondrial proteome and identified several mitochondrial proteins as its putative substrates. These findings identify RBX2-CRL5 as a mitochondrial Ub ligase that controls mitophagy under physiological conditions in a PARKIN-independent, PINK1-dependent manner, thereby regulating cardiac homeostasis.

TEAD1 is crucial for developmental myelination, Remak bundles, and functional regeneration of peripheral nerves.

Previously we showed that the hippo pathway transcriptional effectors, YAP and TAZ, are essential for Schwann cells (SCs) to develop, maintain and regenerate myelin . Although TEAD1 has been implicated as a partner transcription factor, the mechanisms by which it mediates YAP/TAZ regulation of SC myelination are unclear. Here, using conditional and inducible knockout mice, we show that TEAD1 is crucial for SCs to develop and regenerate myelin. It promotes myelination by both positively and negatively regulating SC proliferation, enabling Krox20/Egr2 to upregulate myelin proteins, and upregulating the cholesterol biosynthetic enzymes FDPS and IDI1. We also show stage-dependent redundancy of TEAD1 and that non-myelinating SCs have a unique requirement for TEAD1 to enwrap nociceptive axons in Remak bundles. Our findings establish TEAD1 as a major partner of YAP/TAZ in developmental myelination and functional nerve regeneration and as a novel transcription factor regulating Remak bundle integrity.

TEAD1 is crucial for developmental myelination, Remak bundles, and functional regeneration of peripheral nerves.

Previously we showed that the hippo pathway transcriptional effectors, YAP and TAZ, are essential for Schwann cells (SCs) to develop, maintain and regenerate myelin (Grove et al., 2017; Grove, Lee, Zhao, & Son, 2020). Although TEAD1 has been implicated as a partner transcription factor, the mechanisms by which it mediates YAP/TAZ regulation of SC myelination are unclear. Here, using conditional and inducible knockout mice, we show that TEAD1 is crucial for SCs to develop and regenerate myelin. It promotes myelination by both positively and negatively regulating SC proliferation, enabling Krox20/Egr2 to upregulate myelin proteins, and upregulating the cholesterol biosynthetic enzymes FDPS and IDI1. We also show stage-dependent redundancy of TEAD1 and that non-myelinating SCs have a unique requirement for TEAD1 to enwrap nociceptive axons in Remak bundles. Our findings establish TEAD1 as a major partner of YAP/TAZ in developmental myelination and functional nerve regeneration and as a novel transcription factor regulating Remak bundle integrity.

SMYD2 regulates vascular smooth muscle cell phenotypic switching and intimal hyperplasia via interaction with myocardin.

The SET and MYND domain-containing protein 2 (SMYD2) is a histone lysine methyltransferase that has been reported to regulate carcinogenesis and inflammation. However, its role in vascular smooth muscle cell (VSMC) homeostasis and vascular diseases has not been determined. Here, we investigated the role of SMYD2 in VSMC phenotypic modulation and vascular intimal hyperplasia and elucidated the underlying mechanism. We observed that SMYD2 expression was downregulated in injured carotid arteries in mice and phenotypically modulated VSMCs in vitro. Using an SMC-specific SMYD2 knockout mouse model, we found that SMYD2 ablation in VSMCs exacerbated neointima formation after vascular injury in vivo. Conversely, SMYD2 overexpression inhibited VSMC proliferation and migration in vitro and attenuated arterial narrowing in injured vessels in mice. SMYD2 downregulation promoted VSMC phenotypic switching accompanied with enhanced proliferation and migration. Mechanistically, genome-wide transcriptome analysis and loss/gain-of-function studies revealed that SMYD2 up-regulated VSMC contractile gene expression and suppressed VSMC proliferation and migration, in part, by promoting expression and transactivation of the master transcription cofactor myocardin. In addition, myocardin directly interacted with SMYD2, thereby facilitating SMYD2 recruitment to the CArG regions of SMC contractile gene promoters and leading to an open chromatin status around SMC contractile gene promoters via SMYD2-mediated H3K4 methylation. Hence, we conclude that SMYD2 is a novel regulator of VSMC contractile phenotype and intimal hyperplasia via a myocardin-dependent epigenetic regulatory mechanism.

SMYD2 Regulates Vascular Smooth Muscle Cell Phenotypic Switching and Intimal Hyperplasia via Interaction with Myocardin.

The SET and MYND domain-containing protein 2 (SMYD2) is a histone lysine methyltransferase that has been reported to regulate carcinogenesis and inflammation. However, its role in vascular smooth muscle cell (VSMC) homeostasis and vascular diseases has not been determined. Here, we investigated the role of SMYD2 in VSMC phenotypic modulation and vascular intimal hyperplasia and elucidated the underlying mechanism. We observed that SMYD2 expression was downregulated in injured carotid arteries in mice and phenotypically modulated VSMCs in vitro. Using a SMC-specific Smyd2 knockout mouse model, we found that Smyd2 ablation in VSMCs exacerbates neointima formation after vascular injury in vivo. Conversely, Smyd2 overexpression inhibits VSMC proliferation and migration in vitro and attenuates arterial narrowing in injured vessels in mice. Smyd2 downregulation promotes VSMC phenotypic switching accompanied with enhanced proliferation and migration. Mechanistically, genome-wide transcriptome analysis and loss/gain-of-function studies revealed that SMYD2 up-regulates VSMC contractile gene expression and suppresses VSMC proliferation and migration, in part, by promoting expression and transactivation of the master transcription cofactor myocardin. In addition, myocardin directly interacts with SMYD2, thereby facilitating SMYD2 recruitment to the CArG regions of SMC contractile gene promoters and leading to an open chromatin status around SMC contractile gene promoters via SMYD2-mediated H3K4 methylation. Hence, we conclude that SMYD2 is a novel regulator of VSMC contractile phenotype and intimal hyperplasia via a myocardin-dependent epigenetic regulatory mechanism and may be a potential therapeutic target for occlusive vascular diseases.

Coronary Artery Disease Risk Gene PRDM16 is Preferentially Expressed in Vascular Smooth Muscle Cells and a Potential Novel Regulator of Smooth Muscle Homeostasis.

Objective: Vascular smooth muscle cells (VSMCs) are the primary contractile component of blood vessels and can undergo phenotypic switching from a contractile to a synthetic phenotype in vascular diseases such as coronary artery disease (CAD). This process leads to decreased expression of SMC lineage genes and increased proliferative, migratory and secretory abilities that drive disease progression. Super-enhancers (SE) and occupied transcription factors are believed to drive expression of genes that maintain cell identify and homeostasis. The goal of this study is to identify novel regulator of VSMC homeostasis by screening for SE-regulated transcription factors in arterial tissues. Approach and Results: We characterized human artery SEs by analyzing the enhancer histone mark H3K27ac ChIP-seq data of multiple arterial tissues. We unexpectedly discovered the transcription factor PRDM16, a GWAS identified CAD risk gene with previously well-documented roles in brown adipocytes but with an unknown function in vascular disease progression, is enriched with artery-specific SEs. Further analysis of public bulk RNA-seq and scRNA-seq datasets, as well as qRT-PCR and Western blotting analysis, demonstrated that PRDM16 is preferentially expressed in arterial tissues and in contractile VSMCs but not in visceral SMCs, and down-regulated in phenotypically modulated VSMCs. To explore the function of Prdm16 in vivo, we generated Prdm16 SMC-specific knockout mice and performed histological and bulk RNA-Seq analysis of aortic tissues. SMC-deficiency of Prdm16 does not affect the aortic morphology but significantly alters expression of many CAD risk genes and genes involved in VSMC phenotypic modulation. Specifically, Prdm16 negatively regulates the expression of Tgfb2 that encodes for an upstream ligand of TGF-ß signaling pathway, potentially through binding to the promoter region of Tgfb2 . These transcriptomic changes likely disrupt VSMC homeostasis and predispose VSMCs to a disease state. Conclusions: Our results suggest that the CAD risk gene PRDM16 is preferentially expressed in VSMCs and is a novel regulator of VSMC homeostasis. Future studies are warranted to investigate its role in VSMCs under pathological conditions such as atherosclerosis.

The Long Noncoding RNA Cardiac Mesoderm Enhancer-Associated Noncoding RNA (Carmn) Is a Critical Regulator of Gastrointestinal Smooth Muscle Contractile Function and Motility.

BACKGROUND & AIMS: Visceral smooth muscle cells (SMCs) are an integral component of the gastrointestinal (GI) tract that regulate GI motility. SMC contraction is regulated by posttranslational signaling and the state of differentiation. Impaired SMC contraction is associated with significant morbidity and mortality, but the mechanisms regulating SMC-specific contractile gene expression, including the role of long noncoding RNAs (lncRNAs), remain largely unexplored. Herein, we reveal a critical role of Carmn (cardiac mesoderm enhancer-associated noncoding RNA), an SMC-specific lncRNA, in regulating visceral SMC phenotype and contractility of the GI tract. METHODS: Genotype-Tissue Expression and publicly available single-cell RNA sequencing (scRNA-seq) data sets from embryonic, adult human, and mouse GI tissues were interrogated to identify SMC-specific lncRNAs. The functional role of Carmn was investigated using novel green fluorescent protein (GFP) knock-in (KI) reporter/knock-out (KO) mice. Bulk RNA-seq and single nucleus RNA sequencing (snRNA-seq) of colonic muscularis were used to investigate underlying mechanisms. RESULTS: Unbiased in silico analyses and GFP expression patterns in Carmn GFP KI mice revealed that Carmn is highly expressed in GI SMCs in humans and mice. Premature lethality was observed in global Carmn KO and inducible SMC-specific KO mice due to GI pseudo-obstruction and severe distension of the GI tract, with dysmotility in cecum and colon segments. Histology, GI transit, and muscle myography analysis revealed severe dilation, significantly delayed GI transit, and impaired GI contractility in Carmn KO vs control mice. Bulk RNA-seq of GI muscularis revealed that loss of Carmn promotes SMC phenotypic switching, as evidenced by up-regulation of extracellular matrix genes and down-regulation of SMC contractile genes, including Mylk, a key regulator of SMC contraction. snRNA-seq further revealed SMC Carmn KO not only compromised myogenic motility by reducing contractile gene expression but also impaired neurogenic motility by disrupting cell-cell connectivity in the colonic muscularis. These findings may have translational significance, because silencing CARMN in human colonic SMCs significantly attenuated contractile gene expression, including MYLK, and decreased SMC contractility. Luciferase reporter assays showed that CARMN enhances the transactivation activity of the master regulator of SMC contractile phenotype, myocardin, thereby maintaining the GI SMC myogenic program. CONCLUSIONS: Our data suggest that Carmn is indispensable for maintaining GI SMC contractile function in mice and that loss of function of CARMN may contribute to human visceral myopathy. To our knowledge this is the first study showing an essential role of lncRNA in the regulation of visceral SMC phenotype.

Neddylation is required for perinatal cardiac development through stimulation of metabolic maturation.

Cardiac maturation is crucial for postnatal cardiac development and is increasingly known to be regulated by a series of transcription factors. However, post-translational mechanisms regulating this process remain unclear. Here we report the indispensable role of neddylation in cardiac maturation. Mosaic deletion of NAE1, an essential enzyme for neddylation, in neonatal hearts results in the rapid development of cardiomyopathy and heart failure. NAE1 deficiency disrupts transverse tubule formation, inhibits physiological hypertrophy, and represses fetal-to-adult isoform switching, thus culminating in cardiomyocyte immaturation. Mechanistically, we find that neddylation is needed for the perinatal metabolic transition from glycolytic to oxidative metabolism in cardiomyocytes. Further, we show that HIF1α is a putative neddylation target and that inhibition of neddylation accumulates HIF1α and impairs fatty acid utilization and bioenergetics in cardiomyocytes. Together, our data show neddylation is required for cardiomyocyte maturation through promoting oxidative metabolism in the developing heart.

Hepatic neddylation deficiency triggers fatal liver injury via inducing NF-κB-inducing kinase in mice.

The conjugation of neural precursor cell expressed, developmentally downregulated 8 (NEDD8) to target proteins, termed neddylation, participates in many cellular processes and is aberrant in various pathological diseases. Its relevance to liver function and failure remains poorly understood. Herein, we show dysregulated expression of NAE1, a regulatory subunit of the only NEDD8 E1 enzyme, in human acute liver failure. Embryonic- and adult-onset deletion of NAE1 in hepatocytes causes hepatocyte death, inflammation, and fibrosis, culminating in fatal liver injury in mice. Hepatic neddylation deficiency triggers oxidative stress, mitochondrial dysfunction, and hepatocyte reprogramming, potentiating liver injury. Importantly, NF-κB-inducing kinase (NIK), a serine/Thr kinase, is a neddylation substrate. Neddylation of NIK promotes its ubiquitination and degradation. Inhibition of neddylation conversely causes aberrant NIK activation, accentuating hepatocyte damage and inflammation. Administration of N-acetylcysteine, a glutathione surrogate and antioxidant, mitigates liver failure caused by hepatic NAE1 deletion in adult male mice. Therefore, hepatic neddylation is important in maintaining postnatal and adult liver homeostasis, and the identified neddylation targets/pathways provide insights into therapeutically intervening acute liver failure.

Receptor-independent fluid-phase macropinocytosis promotes arterial foam cell formation and atherosclerosis.

Accumulation of lipid-laden foam cells in the arterial wall plays a central role in atherosclerotic lesion development, plaque progression, and late-stage complications of atherosclerosis. However, there are still fundamental gaps in our knowledge of the underlying mechanisms leading to foam cell formation in atherosclerotic arteries. Here, we investigated the role of receptor-independent macropinocytosis in arterial lipid accumulation and pathogenesis of atherosclerosis. Genetic inhibition of fluid-phase macropinocytosis in myeloid cells (LysMCre+ Nhe1fl/fl) and repurposing of a Food and Drug Administration (FDA)-approved drug that inhibits macrophage macropinocytosis substantially decreased atherosclerotic lesion development in low-density lipoprotein (LDL) receptor-deficient and Apoe-/- mice. Stimulation of macropinocytosis using genetic (H-RASG12V) and physiologically relevant approaches promoted internalization of unmodified native (nLDL) and modified [e.g., acetylated (ac) and oxidized (ox) LDL] lipoproteins in both wild-type and scavenger receptor (SR) knockout (Cd36-/-/Sra-/-) macrophages. Pharmacological inhibition of macropinocytosis in hypercholesterolemic wild-type and Cd36-/-/Sra-/- mice identified an important role of macropinocytosis in LDL uptake by lesional macrophages and development of atherosclerosis. Furthermore, serial section high-resolution imaging, LDL immunolabeling, and three-dimensional (3D) reconstruction of subendothelial foam cells provide visual evidence of lipid macropinocytosis in both human and murine atherosclerotic arteries. Our findings complement the SR paradigm of atherosclerosis and identify a therapeutic strategy to counter the development of atherosclerosis and cardiovascular disease.

lncRNA Neat1 regulates neuronal dysfunction post-sepsis via stabilization of hemoglobin subunit beta.

Sepsis-associated encephalopathy (SAE) is characterized by acute and diffuse brain dysfunction and correlates with long-term cognitive impairments with no targeted therapy. We used a mouse model of sepsis-related cognitive impairment to examine the role of lncRNA nuclear enriched abundant transcript 1 (Neat1) in SAE. We observed that Neat1 expression was increased in neuronal cells from septic mice and that it directly interacts with hemoglobin subunit beta (Hbb), preventing its degradation. The Neat1/Hbb axis suppressed postsynaptic density protein 95 (PSD-95) levels and decreased dendritic spine density. Neat1 knockout mice exhibited decreased Hbb levels, which resulted in increased PSD-95 levels, increased neuronal dendritic spine density, and decreased anxiety and memory impairment. Neat1 silencing via the antisense oligonucleotide GapmeR ameliorated anxiety-like behavior and cognitive impairment post-sepsis. In conclusion, we uncovered a previously unknown mechanism of the Neat1/Hbb axis in regulating neuronal dysfunction, which may lead to a novel treatment strategy for SAE.

Loss of myeloid cell-specific SIRPα, but not CD47, attenuates inflammation and suppresses atherosclerosis.

AIMS: Inhibitors of the anti-phagocytic CD47-SIRPα immune checkpoint are currently in clinical development for a variety of haematological and solid tumours. Application of immune checkpoint inhibitors to the cardiovascular field is limited by the lack of preclinical studies using genetic models of CD47 and SIRPα inhibition. In this study, we comprehensively analysed the effects of global and cell-specific SIRPα and CD47 deletion on atherosclerosis development. METHODS AND RESULTS: Here, we show that both SIRPα and CD47 expression are increased in human atherosclerotic arteries and primarily co-localize to CD68+ areas in the plaque region. Hypercholesterolaemic mice homozygous for a Sirpa mutant lacking the signalling cytoplasmic region (Sirpamut/mut) and myeloid cell-specific Sirpa-knockout mice are protected from atherosclerosis. Further, global Cd47-/- mice are protected from atherosclerosis but myeloid cell-specific deletion of Cd47 increased atherosclerosis development. Using a combination of techniques, we show that loss of SIRPα signalling in macrophages stimulates efferocytosis, reduces cholesterol accumulation, promotes lipid efflux, and attenuates oxidized LDL-induced inflammation in vitro and induces M2 macrophage phenotype and inhibits necrotic core formation in the arterial wall in vivo. Conversely, loss of myeloid cell CD47 inhibited efferocytosis, impaired cholesterol efflux, augmented cellular inflammation, stimulated M1 polarization, and failed to decrease necrotic core area in atherosclerotic vessels. Finally, comprehensive blood cell analysis demonstrated lower haemoglobin and erythrocyte levels in Cd47-/- mice compared with wild-type and Sirpamut/mut mice. CONCLUSION: Taken together, these findings identify SIRPα as a potential target in atherosclerosis and suggest the importance of cell-specific CD47 inhibition as a future therapeutic strategy.

CARMN Is an Evolutionarily Conserved Smooth Muscle Cell-Specific LncRNA That Maintains Contractile Phenotype by Binding Myocardin.

BACKGROUND: Vascular homeostasis is maintained by the differentiated phenotype of vascular smooth muscle cells (VSMCs). The landscape of protein coding genes comprising the transcriptome of differentiated VSMCs has been intensively investigated but many gaps remain including the emerging roles of noncoding genes. METHODS: We reanalyzed large-scale, publicly available bulk and single-cell RNA sequencing datasets from multiple tissues and cell types to identify VSMC-enriched long noncoding RNAs. The in vivo expression pattern of a novel smooth muscle cell (SMC)-expressed long noncoding RNA, Carmn (cardiac mesoderm enhancer-associated noncoding RNA), was investigated using a novel Carmn green fluorescent protein knock-in reporter mouse model. Bioinformatics and quantitative real-time polymerase chain reaction analysis were used to assess CARMN expression changes during VSMC phenotypic modulation in human and murine vascular disease models. In vitro, functional assays were performed by knocking down CARMN with antisense oligonucleotides and overexpressing Carmn by adenovirus in human coronary artery SMCs. Carotid artery injury was performed in SMC-specific Carmn knockout mice to assess neointima formation and the therapeutic potential of reversing CARMN loss was tested in a rat carotid artery balloon injury model. The molecular mechanisms underlying CARMN function were investigated using RNA pull-down, RNA immunoprecipitation, and luciferase reporter assays. RESULTS: We identified CARMN, which was initially annotated as the host gene of the MIR143/145 cluster and recently reported to play a role in cardiac differentiation, as a highly abundant and conserved, SMC-specific long noncoding RNA. Analysis of the Carmn GFP knock-in mouse model confirmed that Carmn is transiently expressed in embryonic cardiomyocytes and thereafter becomes restricted to SMCs. We also found that Carmn is transcribed independently of Mir143/145. CARMN expression is dramatically decreased by vascular disease in humans and murine models and regulates the contractile phenotype of VSMCs in vitro. In vivo, SMC-specific deletion of Carmn significantly exacerbated, whereas overexpression of Carmn markedly attenuated, injury-induced neointima formation in mouse and rat, respectively. Mechanistically, we found that Carmn physically binds to the key transcriptional cofactor myocardin, facilitating its activity and thereby maintaining the contractile phenotype of VSMCs. CONCLUSIONS: CARMN is an evolutionarily conserved SMC-specific long noncoding RNA with a previously unappreciated role in maintaining the contractile phenotype of VSMCs and is the first noncoding RNA discovered to interact with myocardin.

Isolation and reactivity of a gold(I) hydroxytrifluoroborate complex stabilized by anion-π+ interactions.

A 9,9-dimethylxanthene-based ligand substituted at the 4- and 5-positions by a phosphine and a xanthylium unit, respectively, has been prepared and converted into an AuCl complex, the structure of which reveals an intramolecular Au-Cl⋯π+ interaction. This new ligand platform was also found to support the formation of an unprecedented hydroxytrifluoroborate derivative featuring a "hard/soft" mismatched Au-µ(OH)-BF3 motif. Despite its surprising stability, this gold hydroxytrifluoroborate complex is a remarkably potent carbophilic catalyst which readily activates alkynes, without activator.

YAP1/TEAD1 upregulate platelet-derived growth factor receptor beta to promote vascular smooth muscle cell proliferation and neointima formation.

We have previously demonstrated that the transcription co-factor yes-associated protein 1 (YAP1) promotes vascular smooth muscle cell (VSMC) de-differentiation. Yet, the role and underlying mechanisms of YAP1 in neointima formation in vivo remain unclear. The goal of this study was to investigate the role of VSMC-expressed YAP1 in vascular injury-induced VSMC proliferation and delineate the mechanisms underlying its action. Experiments employing gain- or loss-of-function of YAP1 demonstrated that YAP1 promotes human VSMC proliferation. Mechanistically, we identified platelet-derived growth factor receptor beta (PDGFRB) as a novel YAP1 target gene that confers the YAP1-dependent hyper-proliferative effects in VSMCs. Furthermore, we identified TEA domain transcription factor 1 (TEAD1) as a key transcription factor that mediates YAP1-dependent PDGFRß expression. ChIP assays demonstrated that TEAD1 is enriched at a PDGFRB gene enhancer. Luciferase reporter assays further demonstrated that YAP1 and TEAD1 co-operatively activate the PDGFRB enhancer. Consistent with these observations, we found that YAP1 expression is upregulated after arterial injury and correlates with PDGFRß expression and VSMC proliferation in vivo. Using a novel inducible SM-specific Yap1 knockout mouse model, we found that the specific deletion of Yap1 in adult VSMCs is sufficient to attenuate arterial injury-induced neointima formation, largely due to inhibited PDGFRß expression and VSMC proliferation. Our study unravels a novel mechanism by which YAP1/TEAD1 promote VSMC proliferation via transcriptional induction of PDGFRß, thereby enhancing PDGF-BB downstream signaling and promoting neointima formation.

Deficiency of the novel high mobility group protein HMGXB4 protects against systemic inflammation-induced endotoxemia in mice.

Sepsis is a major cause of mortality in intensive care units, which results from a severely dysregulated inflammatory response that ultimately leads to organ failure. While antibiotics can help in the early stages, effective strategies to curtail inflammation remain limited. The high mobility group (HMG) proteins are chromosomal proteins with important roles in regulating gene transcription. While HMGB1 has been shown to play a role in sepsis, the role of other family members including HMGXB4 remains unknown. We found that expression of HMGXB4 is strongly induced in response to lipopolysaccharide (LPS)-elicited inflammation in murine peritoneal macrophages. Genetic deletion of Hmgxb4 protected against LPS-induced lung injury and lethality and cecal ligation and puncture (CLP)-induced lethality in mice, and attenuated LPS-induced proinflammatory gene expression in cultured macrophages. By integrating genome-wide transcriptome profiling and a publicly available ChIP-seq dataset, we identified HMGXB4 as a transcriptional activator that regulates the expression of the proinflammatory gene, Nos2 (inducible nitric oxide synthase 2) by binding to its promoter region, leading to NOS2 induction and excessive NO production and tissue damage. Similar to Hmgxb4 ablation in mice, administration of a pharmacological inhibitor of NOS2 robustly decreased LPS-induced pulmonary vascular permeability and lethality in mice. Additionally, we identified the cell adhesion molecule, ICAM1, as a target of HMGXB4 in endothelial cells that facilitates inflammation by promoting monocyte attachment. In summary, our study reveals a critical role of HMGXB4 in exacerbating endotoxemia via transcriptional induction of Nos2 and Icam1 gene expression and thus targeting HMGXB4 may be an effective therapeutic strategy for the treatment of sepsis.