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Angiogenesis, the formation of new blood vessels, is tightly regulated by gene transcriptional programs. Yin Ying 1 (YY1) is a ubiquitously distributed transcription factor with diverse and complex biological functions; however, little is known about the cell-type-specific role of YY1 in vascular development and angiogenesis. Here we report that endothelial cell (EC)-specific YY1 deletion in mice led to embryonic lethality as a result of abnormal angiogenesis and vascular defects. Tamoxifen-inducible EC-specific YY1 knockout (YY1iΔEC) mice exhibited a scarcity of retinal sprouting angiogenesis with fewer endothelial tip cells. YY1iΔEC mice also displayed severe impairment of retinal vessel maturation. In an ex vivo mouse aortic ring assay and a human EC culture system, YY1 depletion impaired endothelial sprouting and migration. Mechanistically, YY1 functions as a repressor protein of Notch signaling that controls EC tip-stalk fate determination. YY1 deficiency enhanced Notch-dependent gene expression and reduced tip cell formation. Specifically, YY1 bound to the N-terminal domain of RBPJ (recombination signal binding protein for Ig Kappa J region) and competed with the Notch coactivator MAML1 (mastermind-like protein 1) for binding to RBPJ, thereby impairing the NICD (intracellular domain of the Notch protein)/MAML1/RBPJ complex formation. Our study reveals an essential role of endothelial YY1 in controlling sprouting angiogenesis through directly interacting with RBPJ and forming a YY1-RBPJ nuclear repression complex. [ABSTRACT FROM AUTHOR]

SENCR is a human-specific, vascular cell-enriched long-noncoding RNA (lncRNA) that regulates vascular smooth muscle cell and endothelial cell (EC) phenotypes. The underlying mechanisms of action of SENCR in these and other cell types is unknown. Here, levels of SENCR RNA are shown to be elevated in several differentiated human EC lineages subjected to laminar shear stress. Increases in SENCR RNA are also observed in the laminar shear stress region of the adult aorta of humanized SENCR-expressing mice, but not in disturbed shear stress regions. SENCR loss-of-function studies disclose perturbations in EC membrane integrity resulting in increased EC permeability. Biotinylated RNA pull-down and mass spectrometry establish an abundant SENCR-binding protein, cytoskeletal-associated protein 4 (CKAP4); this ribonucleoprotein complex was further confirmed in an RNA immunoprecipitation experiment using an antibody to CKAP4. Structure-function studies demonstrate a noncanonical RNA-binding domain in CKAP4 that binds SENCR. Upon SENCR knockdown, increasing levels of CKAP4 protein are detected in the EC surface fraction. Furthermore, an interaction between CKAP4 and CDH5 is enhanced in SENCR-depleted EC. This heightened association appears to destabilize the CDH5/CTNND1 complex and augment CDH5 internalization, resulting in impaired adherens junctions. These findings support SENCR as a flow-responsive lncRNA that promotes EC adherens junction integrity through physical association with CKAP4, thereby stabilizing cell membrane-bound CDH5. [ABSTRACT FROM AUTHOR]

Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital visceral myopathy characterized by severe dilation of the urinary bladder and defective intestinal motility. The genetic basis of MMIHS has been ascribed to spontaneous and autosomal dominant mutations in actin gamma 2 (ACTG2), a smooth muscle contractile gene. However, evidence suggesting a recessive origin of the disease also exists. Using combined homozygosity mapping and whole exome sequencing, a genetically isolated family was found to carry a premature termination codon in Leiomodin1 (LMOD1), a gene preferentially expressed in vascular and visceral smooth muscle cells. Parents heterozygous for the mutation exhibited no abnormalities, but a child homozygous for the premature termination codon displayed symptoms consistent with MMIHS. We used CRISPR-Cas9 (CRISPR-associated protein) genome editing of Lmod1 to generate a similar premature termination codon. Mice homozygous for the mutation showed loss of LMOD1 protein and pathology consistent with MMIHS, including late gestation expansion of the bladder, hydronephrosis, and rapid demise after parturition. Loss of LMOD1 resulted in a reduction of filamentous actin, elongated cytoskeletal dense bodies, and impaired intestinal smooth muscle contractility. These results define LMOD1 as a disease gene for MMIHS and suggest its role in establishing normal smooth muscle cytoskeletal–contractile coupling. [ABSTRACT FROM AUTHOR]

Dynamic nucleocytoplasmic shuttling of class IIa histone deacetylases (HDACs) is a fundamental mechanism regulating gene transcription. Recent studies have identified several protein kinases that phosphorylate HDAC5, leading to its exportation from the nucleus. However, the negative regulatory mechanisms for HDAC5 nuclear exclusion remain largely unknown. Here we show that cAMP-activated protein kinase A (PKA) specifically phosphorylates HDAC5 and prevents its export from the nucleus, leading to suppression of gene transcription. PKA interacts directly with HDAC5 and phosphorylates HDAC5 at serine 280, an evolutionarily conserved site. Phosphorylation of HDAC5 by PKA interrupts the association of HDAC5 with protein chaperone 14-3-3 and hence inhibits stress signal-induced nuclear export of HDAC5. An HDAC5 mutant that mimics PKA-dependent phosphorylation localizes in the nucleus and acts as a dominant inhibitor for myocyte enhancer factor 2 transcriptional activity. Molecular manipulations of HDAC5 show that PKA-phosphorylated HDAC5 inhibits cardiac fetal gene expression and cardiomyocyte hypertrophy. Our findings identify HDAC5 as a substrate of PKA and reveal a cAMP/PKA-dependent pathway that controls HDAC5 nucleocytoplasmic shuttling and represses gene transcription. This pathway may represent a mechanism by which cAMP/PKA signaling modulates a wide range of biological functions and human diseases such as cardiomyopathy. [ABSTRACT FROM AUTHOR]

X-box binding protein 1 (XBP1) is a key signal transducer' in endoplasmic reticulum stress response, and its potential role in the atherosclerosis development is unknown. This study aims to explore the impact of XBP1 on maintaining endothelial integrity related to atherosclerosis and to delineate the underlying mechanism. We found that XBP1 was highly expressed at branch points and areas of atherosclerotic lesions in the arteries of ApoE-/- mice, which was related to the severity of lesion development. In vitro study using human umbilical vein endothelial cells (HUVECs) indicated that disturbed flow increased the activation of XBP1 expression and splicing. Overexpression of spliced XBP1 induced apoptosis of HUVECs and endothelial loss from blood vessels during ex vivo cultures because of caspase activation and down-regulation of VE-cadherin resulting from transcriptional suppression and matrix metalloproteinase-mediated degradation. Reconstitution of VE-cadherin by Ad-VEcad significantly increased Ad-XBP1s-infected 1-IUVEC survival. Importantly, Ad-XBP1s gene transfer to the vessel wall of ApoE-/- mice resulted in development of atherosclerotic lesions after aorta isografting. These results indicate that XBP1 plays an important role in maintaining endothelial integrity and atherosclerosis development, which provides a potential therapeutic target to intervene in atherosclerosis. [ABSTRACT FROM AUTHOR]