Recently, messenger RNAs in eukaryotes have shown to associate with antisense (AS) transcript partners that are often referred to as long noncoding RNAs (lncRNAs) whose function is largely unknown. Here, we have identified a natural AS transcript for tyrosine kinase containing immunoglobulin and epidermal growth factor homology domain-1 (tie-1), tie-1AS lncRNA in zebrafish, mouse, and humans. In embryonic zebrafish, tie-1AS lncRNA transcript is expressed temporally and spatially in vivo with its native target, the tie-1 coding transcript and in additional locations (ear and brain). The tie-1AS lncRNA selectively binds tie-1 mRNA in vivo and regulates tie-1 transcript levels, resulting in specific defects in endothelial cell con- IntroductionOver the past few years, intensive unbiased analysis of transcriptome species has revealed that eukaryotic genomes contain a variety of RNA species. RNA molecules are essentially classified into 2 types, protein coding and nonprotein coding. The protein-coding transcripts or messenger RNA (mRNA) account for only approximately 2.3% of the human genome. 1 The majority of transcription appears to be nonprotein coding or noncoding, and the function of these noncoding transcripts is largely unknown. 2 Of the noncoding RNAs, the regulatory short noncoding RNAs, such as microRNAs, are well studied. The long noncoding RNAs (lncRNAs), which compose the largest portion of the mammalian noncoding transcriptome, are the least understood, especially its function. 3,4 lncRNAs are oriented in sense or antisense (AS) direction with respect to a protein coding locus, and located in intronic or intergenic regions. 5 In humans and mice, 61% to 72% of all transcribed regions possess lncRNAs in AS orientation, 2,6 and AS lncRNA transcripts play important roles in pathogenesis. For instance, the BACE1-AS transcript was elevated in subjects with Alzheimer disease and in amyloid precursor protein transgenic mice. 7 A growing body of evidence suggests that lncRNAs for most critical physiologic processes will be identified. Angiogenesis, the development of new vasculature from existing vasculature, is one of the fundamental developmental physiologic processes regulated in a developing vertebrate embryo. 8 Here, we identify a natural AS transcript for tyrosine kinase containing immunoglobulin and epidermal growth factor homology domain-1 (tie-1), tie-1AS lncRNA in zebrafish, mouse, and humans. tie-1 is a cell-surface tyrosine kinase receptor for angiopoietin ligands that is known to play a role in vascular development in vertebrates. [9][10][11][12] In embryonic zebrafish, tie-1AS lncRNA transcript is expressed temporally and spatially in vivo with its native target, the tie-1 coding transcript, and in additional locations (ear and brain). Its expression is controlled by a 3-kb genomic fragment in the 3Ј region of tie-1, and the bioinformatic predicted hybrid structure between tie-1:tie-1AS was detected in vivo. Capped or uncapped tie-1AS lncRNA selectively binds tie-1 mRNA in vivo and regulates tie-1...

The antiproliferative effects of gamma-tocotrienol are associated with suppression in epidermal growth factor (EGF)-dependent phosphatidylinositol-3-kinase (PI3K)/PI3K-dependent kinase-1 (PDK-1)/Akt mitogenic signalling in neoplastic mammary epithelial cells. Studies were conducted to investigate the direct effects of gamma-tocotrienol treatment on specific components within the PI3K/PDK-1/Akt mitogenic pathway. +SA cells were grown in culture and maintained in serum-free media containing 10 ng/ml EGF as a mitogen. Treatment with 0-8 microm gamma-tocotrienol resulted in a dose-responsive decrease in the +SA cell growth and a corresponding decrease in phospho-Akt (active) levels. However, gamma-tocotrienol treatment had no direct inhibitory effect on Akt or PI3K enzymatic activity, suggesting that the inhibitory effects of gamma-tocotrienol occur upstream of PI3K, possibly at the level of the EGF-receptor (ErbB1). Additional studies were conducted to determine the effects of gamma-tocotrienol on ErbB receptor activation. Results showed that gamma-tocotrienol treatment had little or no effect on ErbB1 or ErbB2 receptor tyrosine phosphorylation, a prerequisite for substrate interaction and signal transduction, but did cause a significant and progressive decrease in the ErbB3 tyrosine phosphorylation. Because ErbB1 or ErbB2 receptors form heterodimers with the ErbB3 receptor, and ErbB3 heterodimers have been shown to be the most potent activators of PI3K, these findings strongly suggest that the antiproliferative effects of gamma-tocotrienol in neoplastic +SA mouse mammary epithelial cells are mediated by a suppression in ErbB3-receptor tyrosine phosphorylation and subsequent reduction in PI3K/PDK-1/Akt mitogenic signalling.

Rho GTPases play an important and versatile role in several biological processes. In this study, we identified the zebrafish ortholog of the mammalian Rho A guanine exchange factor (GEF), Synectin-binding GEF (Syx), and determined its in vivo function in the zebrafish and the mouse. We found that Syx is expressed specifically in the vasculature of these organisms. Loss-of-function studies in the zebrafish and mouse point to a specific role for Syx in angiogenic sprouting in the developing vascular bed. Importantly, vasculogenesis and angioblast differentiation steps were unaffected in syx-knockdown (KD) zebrafish embryos, and the vascular sprouting defects were partially rescued by the mouse ortholog. Syx KD in vitro impairs vascular endothelial growth factor-A (VEGF-A) induced endothelial cell migration and angiogenesis. We have also uncovered a potential mechanism of endothelial sprout guidance in which Angiomotin (Amot), a component of endothelial cell junctions, plays an additive role with Syx in directing endothelial sprouts. These results identify Syx as an essential contributor to angiogenesis in vivo.

Background: Roundabouts are axon guidance molecules that have recently been identified to play a role in vascular guidance as well. In this study, we have investigated gene knockdown analysis of endothelial Robos, in particular roundabout 4 (robo4), the predominant Robo in endothelial cells using small interfering RNA technology in vitro.

IntroductionThe mitogen-activated protein kinase (MAPK) pathway 1 represent a key signal integration step inside the cell for various extracellular stimuli. 2 At least 4 MAPK families have been identified: the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38, and Erk5. Each MAPK is activated by specific MAPK kinases (Mkks or Meks), which in turn are regulated by Mek kinases (Mekks). To date, several members of the MAPK family, including Erk5 3 and Mekk3, 4 have been implicated in vascular development in vivo. 5 Furthermore, MAPKs are often associated with pathologic responses, such as stress, apoptosis, inflammation, and cell proliferation. 5,6 In terms of vascular conditions, differential expression of active MAPKs was noticed in several types of hemangiomas, 7 the most common vascular tumor of infancy and childhood, 8 and the presence of immunoreactive phosphorylated MAPK inversely correlate with degree of malignancy. 7 Therefore, from a drug discovery standpoint, vascular specific components of MAPKs cascade are promising drug targets. 6 MAPKs are activated by dual phosphorylation of tyrosine and threonine residues in their activation loops. To control MAPK activation, a family of proteins called dual-specificity protein tyrosine phosphatases (DUSPs) dephosphorylate both residues. 9 DUSPs come in 2 varieties: dual-specificity MAPK phosphatases and atypical DUSPs. A dual specific phosphatase, Dusp-5, was recently identified by 2 independent microarray studies as a vascular-specific gene, 10,11 and we hypothesized that dusp-5 plays a specific role in vascular development. To investigate dusp-5 function, we initiated a loss-of-function (LOF) study in zebrafish (ZF). We identified that dusp-5 LOF embryos showed enhanced etsrp ϩ angioblasts at the lateral plate mesoderm (LPM). Recently, we had identified in a gain-of-function (GOF) and LOF study for a serine-threonine kinase member of the sucrose nonfermenting kinase family, Snrk-1, that it increased or decreased, respectively, the same etsrp ϩ angioblast population at the LPM. 12 Taking these findings together, we hypothesized that, during vascular development, Dusp-5 and Snrk-1 function together in controlling angioblast numbers at the LPM. In this study, we show that dusp-5 is expressed in angioblasts in the embryonic ZF and is essential for vascular development in vivo. We show that loss of Dusp-5 function in vitro causes apoptosis of endothelial cells (ECs), and Dusp-5 and Snrk-1 target a common signaling pathway responsible for maintaining angioblast populations along the LPM. Snrk-1 ectopically induces etsrp ϩ angioblasts in the LPM, which is blocked by Dusp-5 that functions downstream of Snrk-1. In addition, we have identified mutations in dusp-5 and snrk-1 in the lesional tissue of many vascular anomaly patient samples, suggesting a critical role for this pathway in disease. Methods ZF stocksWild-type ZF (TuAB strain) were grown and maintained at 28.5°C. 13 All procedures were performed according to animal protoc...

In vertebrates, molecular mechanisms dictate angioblasts' migration and subsequent differentiation into arteries and veins. In this study, we used a microarray screen to identify a novel member of the sucrose nonfermenting related kinase

Treatment with 4 micromgamma-tocotrienol significantly inhibited +SA cell proliferation over a 4-day culture period. Moreover, this treatment resulted in a relatively large reduction in cyclin D1, cyclin dependent kinase (CDK)4, CDK2 and CDK6 levels, between 4 and 24 h after EGF exposure. Tocotrienol treatment also resulted in a relatively large increase in CDK inhibitor (CKI) p27, prior to and after EGF exposure, but had little effect on levels of CKIs, p21 and p15. Tocotrienol treatment also induced a large relative reduction in retinoblastoma (Rb) protein phosphorylation at ser780 and ser807/811. These findings strongly suggest that anti-proliferative effects of gamma-tocotrienol are associated with reduction in cell cycle progression from G(1) to S, as evidenced by increased p27 levels, and a corresponding decrease in cyclin D1, CDK2, CDK4, CDK6 and phosphorylated Rb levels.

Endothelial cell-specific chemotaxis receptor (ECSCR) is a cell surface protein expressed by blood endothelial cells with roles in endothelial cell migration and signal transduction. We investigated the function of ecscr in the development of the zebrafish vasculature. Zebrafish ecscr is expressed in angioblasts and in axial vessels during angioblast migration and vasculogenesis. Morpholino-directed ecscr knockdown resulted in defective angioblast migration in the posterior lateral plate mesoderm, a process known to depend on vascular endothelial-derived growth factor (VEGF). In cultured cells, transfected ECSCR localized to actin-rich membrane protrusions, colocalizing with kinase insert domain protein receptor (KDR)/VEGF receptor 2 in these regions. ECSCR-silenced cells show reduced VEGF-induced phosphorylation of KDR but not of FMS-like tyrosine kinase 1 (FLT1)/VEGF receptor 1. Finally, chemical inhibition of VEGF receptor activity in zebrafish resulted in angioblast deficiencies that partially overlap with those seen in ecscr morphants. We propose that ecscr promotes migration of zebrafish angioblasts by enhancing endothelial kdr sensitivity to VEGF.