Lucy Liaw

Osteopontin: A Multifunctional Molecule Regulating Chronic Inflammation and Vascular Disease

Osteopontin (OPN) is a multifunctional molecule highly expressed in chronic inflammatory and autoimmune diseases, and it is specifically localized in and around inflammatory cells. OPN is a secreted adhesive molecule, and it is thought to aid in the recruitment of monocytes-macrophages and to regulate cytokine production in macrophages, dendritic cells, and T-cells. OPN has been classified as T-helper 1 cytokine and thus believed to exacerbate inflammation in several chronic inflammatory diseases, including atherosclerosis. Besides proinflammatory functions, physiologically OPN is a potent inhibitor of mineralization, it prevents ectopic calcium deposits and is a potent inducible inhibitor of vascular calcification. Clinically, OPN plasma levels have been found associated with various inflammatory diseases, including cardiovascular burden. It is thus imperative to dissect the OPN proinflammatory and anticalcific functions. OPN recruitment functions of inflammatory cells are thought to be mediated through its adhesive domains, especially the arginine-glycine-aspartate (RGD) sequence that interacts with several integrin heterodimers. However, the integrin receptors and intracellular pathways mediating OPN effects on immune cells are not well established. Furthermore, several studies show that OPN is cleaved by at least 2 classes of proteases: thrombin and matrix-metalloproteases (MMPs). Most importantly, at least in vitro, fragments generated by cleavage not only maintain OPN adhesive functions but also expose new active domains that may impart new activities. The role for OPN proteolytic fragments in vivo is almost completely unexplored. We believe that further knowledge of the effects of OPN fragments on cell responses might help in designing therapeutics targeting inflammatory and cardiovascular diseases.

Osteopontin inhibits mineral deposition and promotes regression of ectopic calcification.

Ectopic calcification, the abnormal calcification of soft tissues, can have severe clinical consequences especially when localized to vital organs such as heart valves, arteries, and kidneys. Recent observations suggest that ectopic calcification, like bone biomineralization, is an actively regulated process. These observations have led a search for molecular determinants of ectopic calcification. A candidate molecule is osteopontin (OPN), a secreted phosphoprotein invariantly associated with both normal and pathological mineral deposits. In the present study, OPN was found to be a natural inhibitor of ectopic calcification in vivo. Glutaraldehyde-fixed aortic valve leaflets showed accelerated and fourfold to fivefold greater calcification after subcutaneous implantation into OPN-null mice compared to wild-type mice. In vitro and in vivo studies suggest that OPN not only inhibits mineral deposition but also actively promotes its dissolution by physically blocking hydroxyapatite crystal growth and inducing expression of carbonic anhydrase II in monocytic cells and promoting acidification of the extracellular milieu. These findings suggest a novel mechanism of OPN action and potential therapeutic approach to the treatment of ectopic calcification.

Inactivation of the Osteopontin Gene Enhances Vascular Calcification of Matrix Gla Protein–deficient Mice: Evidence for Osteopontin as an Inducible Inhibitor of Vascular Calcification In Vivo

Osteopontin (OPN) is abundantly expressed in human calcified arteries. To examine the role of OPN in vascular calcification, OPN mutant mice were crossed with matrix Gla protein (MGP) mutant mice. Mice deficient in MGP alone (MGP−/− OPN+/+) showed calcification of their arteries as early as 2 weeks (wk) after birth (0.33 ± 0.01 mmol/g dry weight), and the expression of OPN in the calcified arteries was greatly up-regulated compared with MGP wild-types. OPN accumulated adjacent to the mineral and colocalized to surrounding cells in the calcified media. Cells synthesizing OPN lacked smooth muscle (SM) lineage markers, SM α-actin and SM22α. However, most of them were not macrophages. Importantly, mice deficient in both MGP and OPN had twice as much arterial calcification as MGP−/− OPN+/+ at 2 wk, and over 3 times as much at 4 wk, suggesting an inhibitory effect of OPN in vascular calcification. Moreover, these mice died significantly earlier (4.4 ± 0.2 wk) than MGP−/− OPN+/+ counterparts (6.6 ± 1.0 wk). The cause of death in these animals was found to be vascular rupture followed by hemorrhage, most likely due to enhanced calcification. These studies are the first to demonstrate a role for OPN as an inducible inhibitor of ectopic calcification in vivo.

Targeted Genome Modification in Mice Using Zinc Finger Nucleases

Homologous recombination-based gene targeting using Mus musculus embryonic stem cells has greatly impacted biomedical research. This study presents a powerful new technology for more efficient and less time-consuming gene targeting in mice using embryonic injection of zinc-finger nucleases (ZFNs), which generate site-specific double strand breaks, leading to insertions or deletions via DNA repair by the nonhomologous end joining pathway. Three individual genes, multidrug resistant 1a (Mdr1a), jagged 1 (Jag1), and notch homolog 3 (Notch3), were targeted in FVB/N and C57BL/6 mice. Injection of ZFNs resulted in a range of specific gene deletions, from several nucleotides to >1000 bp in length, among 20–75% of live births. Modified alleles were efficiently transmitted through the germline, and animals homozygous for targeted modifications were obtained in as little as 4 months. In addition, the technology can be adapted to any genetic background, eliminating the need for generations of backcrossing to achieve congenic animals. We also validated the functional disruption of Mdr1a and demonstrated that the ZFN-mediated modifications lead to true knockouts. We conclude that ZFN technology is an efficient and convenient alternative to conventional gene targeting and will greatly facilitate the rapid creation of mouse models and functional genomics research.

Osteopontin Is a Critical Inhibitor of Calcium Oxalate Crystal Formation and Retention in Renal Tubules

Calcium nephrolithiasis is the most common form of renal stone disease, with calcium oxalate (CaOx) being the predominant constituent of renal stones. Current in vitro evidence implicates osteopontin (OPN) as one of several macromolecular inhibitors of urinary crystallization with potentially important actions at several stages of CaOx crystal formation and retention. To determine the importance of OPN in vivo, hyperoxaluria was induced in mice targeted for the deletion of the OPN gene together with wild-type control mice. Both groups were given 1% ethylene glycol, an oxalate precursor, in their drinking water for up to 4 wk. At 4 wk, OPN-deficient mice demonstrated significant intratubular deposits of CaOx crystals, whereas wild-type mice were completely unaffected. Retained crystals in tissue sections were positively identified as CaOx monohydrate by both polarized optical microscopy and x-ray powder diffraction analysis. Furthermore, hyperoxaluria in the OPN wild-type mice was associated with a significant 2- to 4-fold upregulation of renal OPN expression by immunocytochemistry, lending further support to a renoprotective role for OPN. These data indicate that OPN plays a critical renoprotective role in vivo as an inhibitor of CaOx crystal formation and retention in renal tubules.

Members of the Jagged/Notch Gene Families Are Expressed in Injured Arteries and Regulate Cell Phenotype via Alterations in Cell Matrix and Cell-Cell Interaction

The Jagged/Notch signaling pathways control cell fate determination and differentiation, and their dysfunction is associated with human pathologies involving cardiovascular abnormalities. To determine the presence of these genes during vascular response to injury, we analyzed expression of Jagged1, Jagged2, and Notch1 through 4 after balloon catheter denudation of the rat carotid artery. Although low levels of Jagged1, Jagged2, and constitutive expression of Notch1 were seen in uninjured endothelium, expression of all was significantly increased in injured vascular cells. High Jagged1 expression was restricted to the regenerating endothelial wound edge, whereas Notch transcripts were abundant in endothelial and smooth muscle cells. To understand the basis for Jagged/Notch control of cellular phenotype, we studied an in vitro model of NIH3T3 cells transfected with a secreted form of the extracellular domain of Jagged1. We report that the soluble Jagged1 protein caused decreased cell-matrix adhesion and cell migration defects. Cadherin-mediated intercellular junctions as well as focal adhesions were modified in soluble Jagged1 transfectants, demonstrating that cell-cell contacts and adhesion plaques may be targets of Jagged/Notch activity. We suggest that Jagged regulation of cell-cell and cell-matrix interactions may contribute to the control of cell migration in situations of tissue remodeling in vivo.

Common features of microRNA target prediction tools

The human genome encodes for over 1800 microRNAs (miRNAs), which are short non-coding RNA molecules that function to regulate gene expression post-transcriptionally. Due to the potential for one miRNA to target multiple gene transcripts, miRNAs are recognized as a major mechanism to regulate gene expression and mRNA translation. Computational prediction of miRNA targets is a critical initial step in identifying miRNA:mRNA target interactions for experimental validation. The available tools for miRNA target prediction encompass a range of different computational approaches, from the modeling of physical interactions to the incorporation of machine learning. This review provides an overview of the major computational approaches to miRNA target prediction. Our discussion highlights three tools for their ease of use, reliance on relatively updated versions of miRBase, and range of capabilities, and these are DIANA-microT-CDS, miRanda-mirSVR, and TargetScan. In comparison across all miRNA target prediction tools, four main aspects of the miRNA:mRNA target interaction emerge as common features on which most target prediction is based: seed match, conservation, free energy, and site accessibility. This review explains these features and identifies how they are incorporated into currently available target prediction tools. MiRNA target prediction is a dynamic field with increasing attention on development of new analysis tools. This review attempts to provide a comprehensive assessment of these tools in a manner that is accessible across disciplines. Understanding the basis of these prediction methodologies will aid in user selection of the appropriate tools and interpretation of the tool output.

Collagen Triple Helix Repeat Containing 1, a Novel Secreted Protein in Injured and Diseased Arteries, Inhibits Collagen Expression and Promotes Cell Migration

Collagen triple helix repeat containing 1 (Cthrc1) was identified in a screen for differentially expressed sequences in balloon-injured versus normal arteries. Cthrc1 expression was not detectable in normal arteries. However, on injury it was transiently expressed by fibroblasts of the remodeling adventitia and by smooth muscle cells of the neointima. It was also found in the matrix of calcifying human atherosclerotic plaques. CTHRC1 is a secreted 28-kDa protein that is glycosylated and highly conserved from lower chordates to mammals. A short collagen motif with 12 Gly-X-Y repeats appears to be responsible for trimerization of the protein and this renders the molecule susceptible to cleavage by collagenase. Cthrc1 mRNA expression levels are increased in response to transforming growth factor-&bgr; and bone morphogenetic protein-4. Cell migration assays performed with CTHRC1-overexpressing fibroblasts and smooth muscle cells demonstrate that increased CTHRC1 levels are associated with enhanced migratory ability. Furthermore, CTHRC1 overexpression caused a dramatic reduction in collagen type I mRNA and protein levels. Our data indicate that the novel molecule CTHRC1 is transiently expressed in the arterial wall in response to injury where it may contribute to vascular remodeling by limiting collagen matrix deposition and promoting cell migration.

Selective expression of an aP2/Fatty Acid Binding Protein4-Cre transgene in non-adipogenic tissues during embryonic development

Mouse strains expressing the site-specific Cre recombinase facilitate conditional ablation or activation of genomic sequences when one or several exons of a gene of interest are flanked by loxP sites. Recently, several strains targeting Cre expression to adipocytes have been developed using promoter sequences from the aP2 (Fatty Acid Binding Protein 4, FABP4) gene for adipose tissue-specific gene expression studies. aP2/FABP4 is predominantly expressed in adipose tissue, and while this promoter provides adipocyte-restricted expression postnatally, its expression throughout embryonic development had not been previously characterized. In this report, we demonstrate that the aP2-Cre transgene is expressed and consistently localized within the embryo from mid-gestation stage 9.5 dpc. By 15.5 dpc, β-gal activity was detected primarily in the brown adipose tissue, trigeminal ganglia, dorsal root ganglia, cartilage primordia and vertebrae. Immunofluorescence staining for Cre recombinase and FABP4 protein showed a corresponding staining pattern similar to that of β-gal, confirming that Cre recombinase was produced in the transgenic line at late stages of development, and overlapped with endogenous aP2/FABP4 production. Further, fat-specific oil red O staining of tissue sections validated the presence of lipids in the stained tissues indicating that adipocytes and/or adipocyte-like cells were indeed present in these tissues. This is the first report to our knowledge to describe and confirm aP2/FABP4 promoter expression in this transgenic line during development in the mouse embryo and indicates that aP2/FABP4 expression occurs not only in mature adipocytes, but has a wider embryonic expression pattern than previously appreciated.

Notch and Transforming Growth Factor-β (TGFβ) Signaling Pathways Cooperatively Regulate Vascular Smooth Muscle Cell Differentiation

Notch and transforming growth factor-β (TGFβ) play pivotal roles during vascular development and the pathogenesis of vascular disease. The interaction of these two pathways is not fully understood. The present study utilized primary human smooth muscle cells (SMC) to examine molecular cross-talk between TGFβ1 and Notch signaling on contractile gene expression. Activation of Notch signaling using Notch intracellular domain or Jagged1 ligand induced smooth muscle α-actin (SM actin), smooth muscle myosin heavy chain, and calponin1, and the expression of Notch downstream effectors hairy-related transcription factors. Similarly, TGFβ1 treatment of human aortic smooth muscle cells induced SM actin, calponin1, and smooth muscle protein 22-α (SM22α) in a dose- and time-dependent manner. Hairy-related transcription factor proteins, which antagonize Notch activity, also suppressed the TGFβ1-induced increase in SMC markers, suggesting a general mechanism of inhibition. We found that Notch and TGFβ1 cooperatively activate SMC marker transcripts and protein through parallel signaling axes. Although the intracellular domain of Notch4 interacted with phosphoSmad2/3 in SMC, this interaction was not observed with Notch1 or Notch2. However, we found that CBF1 co-immunoprecipitated with phosphoSmad2/3, suggesting a mechanism to link canonical Notch signaling to phosphoSmad activity. Indeed, the combination of Notch activation and TGFβ1 treatment led to synergistic activation of a TGFβ-responsive promoter. This increase corresponded to increased levels of phosphoSmad2/3 interaction at Smad consensus binding sites within the SM actin, calponin1, and SM22α promoters. Thus, Notch and TGFβ coordinately induce a molecular and functional contractile phenotype by co-regulation of Smad activity at SMC promoters.