Piezo1 integration of vascular architecture with physiological force

The mechanisms by which physical forces regulate endothelial cells to determine the complexities of vascular structure and function are enigmatic¹⁻⁵. Studies of sensory neurons have suggested Piezo proteins as subunits of Ca²⁺-permeable non-selective cationic channels for detection of noxious mechanical impact⁶⁻⁸. Here we show Piezo1 (Fam38a) channels as sensors of frictional force (shear stress) and determinants of vascular structure in both development and adult physiology. Global or endothelial-specific disruption of mouse Piezo1 profoundly disturbed the developing vasculature and was embryonic lethal within days of the heart beating. Haploinsufficiency was not lethal but endothelial abnormality was detected in mature vessels. The importance of Piezo1 channels as sensors of blood flow was shown by Piezo1 dependence of shear-stress-evoked ionic current and calcium influx in endothelial cells and the ability of exogenous Piezo1 to confer sensitivity to shear stress on otherwise resistant cells. Downstream of this calcium influx there was protease activation and spatial reorganization of endothelial cells to the polarity of the applied force. The data suggest that Piezo1 channels function as pivotal integrators in vascular biology

NUDT2 Disruption Elevates Diadenosine Tetraphosphate (Ap4A) and Down-Regulates Immune Response and Cancer Promotion Genes.

Regulation of gene expression is one of several roles proposed for the stress-induced nucleotide diadenosine tetraphosphate (Ap4A). We have examined this directly by a comparative RNA-Seq analysis of KBM-7 chronic myelogenous leukemia cells and KBM-7 cells in which the NUDT2 Ap4A hydrolase gene had been disrupted (NuKO cells), causing a 175-fold increase in intracellular Ap4A. 6,288 differentially expressed genes were identified with P < 0.05. Of these, 980 were up-regulated and 705 down-regulated in NuKO cells with a fold-change ≥ 2. Ingenuity® Pathway Analysis (IPA®) was used to assign these genes to known canonical pathways and functional networks. Pathways associated with interferon responses, pattern recognition receptors and inflammation scored highly in the down-regulated set of genes while functions associated with MHC class II antigens were prominent among the up-regulated genes, which otherwise showed little organization into major functional gene sets. Tryptophan catabolism was also strongly down-regulated as were numerous genes known to be involved in tumor promotion in other systems, with roles in the epithelial-mesenchymal transition, proliferation, invasion and metastasis. Conversely, some pro-apoptotic genes were up-regulated. Major upstream factors predicted by IPA® for gene down-regulation included NFκB, STAT1/2, IRF3/4 and SP1 but no major factors controlling gene up-regulation were identified. Potential mechanisms for gene regulation mediated by Ap4A and/or NUDT2 disruption include binding of Ap4A to the HINT1 co-repressor, autocrine activation of purinoceptors by Ap4A, chromatin remodeling, effects of NUDT2 loss on transcript stability, and inhibition of ATP-dependent regulatory factors such as protein kinases by Ap4A. Existing evidence favors the last of these as the most probable mechanism. Regardless, our results suggest that the NUDT2 protein could be a novel cancer chemotherapeutic target, with its inhibition potentially exerting strong anti-tumor effects via multiple pathways involving metastasis, invasion, immunosuppression and apoptosis

Proteomic Analysis of Pathways Involved in Estrogen-Induced Growth and Apoptosis of Breast Cancer Cells

Estrogen is a known growth promoter for estrogen receptor (ER)-positive breast cancer cells. Paradoxically, in breast cancer cells that have been chronically deprived of estrogen stimulation, re-introduction of the hormone can induce apoptosis.Here, we sought to identify signaling networks that are triggered by estradiol (E2) in isogenic MCF-7 breast cancer cells that undergo apoptosis (MCF-7:5C) versus cells that proliferate upon exposure to E2 (MCF-7). The nuclear receptor co-activator AIB1 (Amplified in Breast Cancer-1) is known to be rate-limiting for E2-induced cell survival responses in MCF-7 cells and was found here to also be required for the induction of apoptosis by E2 in the MCF-7:5C cells. Proteins that interact with AIB1 as well as complexes that contain tyrosine phosphorylated proteins were isolated by immunoprecipitation and identified by mass spectrometry (MS) at baseline and after a brief exposure to E2 for two hours. Bioinformatic network analyses of the identified protein interactions were then used to analyze E2 signaling pathways that trigger apoptosis versus survival. Comparison of MS data with a computationally-predicted AIB1 interaction network showed that 26 proteins identified in this study are within this network, and are involved in signal transduction, transcription, cell cycle regulation and protein degradation.G-protein-coupled receptors, PI3 kinase, Wnt and Notch signaling pathways were most strongly associated with E2-induced proliferation or apoptosis and are integrated here into a global AIB1 signaling network that controls qualitatively distinct responses to estrogen

Antisense-Mediated Knockdown of NaV1.8, but Not NaV1.9, Generates Inhibitory Effects on Complete Freund's Adjuvant-Induced Inflammatory Pain in Rat

Tetrodotoxin-resistant (TTX-R) sodium channels NaV1.8 and NaV1.9 in sensory neurons were known as key pain modulators. Comparing with the widely reported NaV1.8, roles of NaV1.9 on inflammatory pain are poorly studied by antisense-induced specific gene knockdown. Here, we used molecular, electrophysiological and behavioral methods to examine the effects of antisense oligodeoxynucleotide (AS ODN) targeting NaV1.8 and NaV1.9 on inflammatory pain. Following complete Freund's adjuvant (CFA) inflammation treatment, NaV1.8 and NaV1.9 in rat dorsal root ganglion (DRG) up-regulated mRNA and protein expressions and increased sodium current densities. Immunohistochemical data demonstrated that NaV1.8 mainly localized in medium and small-sized DRG neurons, whereas NaV1.9 only expressed in small-sized DRG neurons. Intrathecal (i.t.) delivery of AS ODN was used to down-regulate NaV1.8 or NaV1.9 expressions confirmed by immunohistochemistry and western blot. Unexpectedly, behavioral tests showed that only NaV1.8 AS ODN, but not NaV1.9 AS ODN could reverse CFA-induced heat and mechanical hypersensitivity. Our data indicated that TTX-R sodium channels NaV1.8 and NaV1.9 in primary sensory neurons played distinct roles in CFA-induced inflammatory pain and suggested that antisense oligodeoxynucleotide-mediated blocking of key pain modulator might point toward a potential treatment strategy against certain types of inflammatory pain

Crystal Structure of Diedel, a Marker of the Immune Response of Drosophila melanogaster

Background: The Drosophila melanogaster gene CG11501 is up regulated after a septic injury and was proposed to act as a negative regulator of the JAK/STAT signaling pathway. Diedel, the CG11501 gene product, is a small protein of 115 residues with 10 cysteines. Methodology/Principal Findings: We have produced Diedel in Drosophila S2 cells as an extra cellular protein thanks to its own signal peptide and solved its crystal structure at 1.15 A ˚ resolution by SIRAS using an iodo derivative. Diedel is composed of two sub domains SD1 and SD2. SD1 is made of an antiparallel b-sheet covered by an a-helix and displays a ferredoxin-like fold. SD2 reveals a new protein fold made of loops connected by four disulfide bridges. Further structural analysis identified conserved hydrophobic residues on the surface of Diedel that may constitute a potential binding site. The existence of two conformations, cis and trans, for the proline 52 may be of interest as prolyl peptidyl isomerisation has been shown to play a role in several physiological mechanisms. The genome of D. melanogaster contains two other genes coding for proteins homologous to Diedel, namely CG43228 and CG34329. Strikingly, apart from Drosophila and the pea aphid Acyrthosiphon pisum, Diedel-related sequences were exclusively identified in a few insect DNA viruses of the Baculoviridae and Ascoviridae families. Conclusion/Significance: Diedel, a marker of the Drosophila antimicrobial/antiviral response, is a member of a small famil

Genetic Evidence for Involvement of Neuronally Expressed S1P1 Receptor in Nociceptor Sensitization and Inflammatory Pain

Sphingosine-1-phosphate (S1P) is a key regulator of immune response. Immune cells, epithelia and blood cells generate high levels of S1P in inflamed tissue. However, it is not known if S1P acts on the endings of nociceptive neurons, thereby contributing to the generation of inflammatory pain. We found that the S1P1 receptor for S1P is expressed in subpopulations of sensory neurons including nociceptors. Both S1P and agonists at the S1P1 receptor induced hypersensitivity to noxious thermal stimulation in vitro and in vivo. S1P-induced hypersensitivity was strongly attenuated in mice lacking TRPV1 channels. S1P and inflammation-induced hypersensitivity was significantly reduced in mice with a conditional nociceptor-specific deletion of the S1P1 receptor. Our data show that neuronally expressed S1P1 receptors play a significant role in regulating nociceptor function and that S1P/S1P1 signaling may be a key player in the onset of thermal hypersensitivity and hyperalgesia associated with inflammation

β-Catenin Loss in Hepatocytes Promotes Hepatocellular Cancer after Diethylnitrosamine and Phenobarbital Administration to Mice

Hepatocellular Carcinoma (HCC) is the fifth most common cancer worldwide. β-Catenin, the central orchestrator of the canonical Wnt pathway and a known oncogene is paramount in HCC pathogenesis. Administration of phenobarbital (PB) containing water (0.05% w/v) as tumor promoter following initial injected intraperitoneal (IP) diethylnitrosamine (DEN) injection (5 µg/gm body weight) as a tumor inducer is commonly used model to study HCC in mice. Herein, nine fifteen-day male β-catenin knockout mice (KO) and fifteen wild-type littermate controls (WT) underwent DEN/PB treatment and were examined for hepatic tumorigenesis at eight months. Paradoxically, a significantly higher tumor burden was observed in KO (p<0.05). Tumors in KO were β-catenin and glutamine synthetase negative and HGF/Met, EGFR & IGFR signaling was unremarkable. A significant increase in PDGFRα and its ligand PDGF-CC leading to increased phosphotyrosine-720-PDGFRα was observed in tumor-bearing KO mice (p<0.05). Simultaneously, these livers displayed increased cell death, stellate cell activation, hepatic fibrosis and cell proliferation. Further, PDGF-CC significantly induced hepatoma cell proliferation especially following β-catenin suppression. Our studies also demonstrate that the utilized DEN/PB protocol in the WT C57BL/6 mice did not select for β-catenin gene mutations during hepatocarcinogenesis. Thus, DEN/PB enhanced HCC in mice lacking β-catenin in the liver may be due to their ineptness at regulating cell survival, leading to enhanced fibrosis and regeneration through PDGFRα activation. β-Catenin downregulation also made hepatoma cells more sensitive to receptor tyrosine kinases and thus may be exploited for therapeutics

Down-regulation of SFRP1 as a putative tumor suppressor gene can contribute to human hepatocellular carcinoma

<p>Abstract</p> <p>Background</p> <p>Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. SFRP1 (the secreted frizzled-related protein 1), a putative tumor suppressor gene mapped onto chromosome 8p12-p11.1, the frequent loss of heterozygosity (LOH) region in human HCC, encodes a Wingless-type (Wnt) signaling antagonist and is frequently inactivated by promoter methylation in many human cancers. However, whether the down-regulation of SFRP1 can contribute to hepatocarcinogenesis still remains unclear.</p> <p>Methods</p> <p>We investigated the expression of SFRP1 through real time RT-PCR and immunohistochemistry staining. The cell growth and colony formation were observed as the overexpression and knockdown of SFRP1. The DNA methylation status within SFRP1 promoter was analyzed through methylation-specific PCR or bisulphate-treated DNA sequencing assays. Loss of heterozygosity was here detected with microsatellite markers.</p> <p>Results</p> <p>SFRP1 was significantly down-regulated in 76.1% (35/46) HCC specimens at mRNA level and in 30% (30/100) HCCs indicated by immunohistochemistry staining, as compared to adjacent non-cancerous livers. The overexpression of SFRP1 can significantly inhibit the cell growth and colony formation of YY-8103, SMMC7721, and Hep3B cells. The RNA interference against the constitutional SFRP1 in the offspring SMMC7721 cells, which were stably transfected by ectopic SFRP1, can markedly promote cell growth of these cells. LOH of both microsatellite markers D8S532 and D8SAC016868 flanking the gene locus was found in 13% (6 of 46 HCCs) and 6.5% (3 of 46 HCCs) of the informative cases, respectively, where 5 of 8 HCC specimens with LOH showed the down-regulation of SFRP1. DNA hypermethylation within SFRP1 promoter was identified in two of three HCC specimens without SFRP1 expression. Moreover, the DNA methylation of SFRP1 promoter was significantly reduced, along with the re-expression of the gene, in those HCC cell lines, Bel7404, QGY7701, and MHCC-H, as treated by DAC.</p> <p>Conclusion</p> <p>Our data suggested that the down-regulation of SFRP1 as a candidate tumor suppressor gene, triggered by the epigenetic and/or genetic events, could contribute to the oncogenesis of HCC.</p

Current cardiac imaging techniques for detection of left ventricular mass

Estimation of left ventricular (LV) mass has both prognostic and therapeutic value independent of traditional risk factors. Unfortunately, LV mass evaluation has been underestimated in clinical practice. Assessment of LV mass can be performed by a number of imaging modalities. Despite inherent limitations, conventional echocardiography has fundamentally been established as most widely used diagnostic tool. 3-dimensional echocardiography (3DE) is now feasible, fast and accurate for LV mass evaluation. 3DE is also superior to conventional echocardiography in terms of LV mass assessment, especially in patients with abnormal LV geometry. Cardiovascular magnetic resonance (CMR) and cardiovascular computed tomography (CCT) are currently performed for LV mass assessment and also do not depend on cardiac geometry and display 3-dimensional data, as well. Therefore, CMR is being increasingly employed and is at the present standard of reference in the clinical setting. Although each method demonstrates advantages over another, there are also disadvantages to receive attention. Diagnostic accuracy of methods will also be increased with the introduction of more advanced systems. It is also likely that in the coming years new and more accurate diagnostic tests will become available. In particular, CMR and CCT have been intersecting hot topic between cardiology and radiology clinics. Thus, good communication and collaboration between two specialties is required for selection of an appropriate test