Victoria L. Heath

Anticancer strategies involving the vasculature.

The growth and metastasis of solid tumors critically depends on their ability to develop their own blood supply, a process known as tumor angiogenesis. Over the past decade much work has been performed to understand this process, and modifying this process provides a key point of therapeutic intervention in the fight against cancer. This Review explores the development of anti-VEGF-based antiangiogenic therapies, of which there are currently three licensed for clinical use worldwide. Although originally anticipated to inhibit the growth of tumor vessels, the induction of vascular normalization caused by these approved agents has provided a novel means of effective delivery of known chemotherapeutic agents. The development of small molecules that target VEGF receptors has resulted in the generation of inhibitors with not only vascular activity but antitumor activity in certain cancers. This Review will address the current status of vascular-disrupting strategies, such as therapies designed to induce tumor collapse by selectively destroying existing tumor vessels. These therapies can be broadly divided into small-molecular-weight vascular-disrupting agents and ligand-directed approaches. We discuss the current status of development, drug mechanisms of actions, combination with conventional chemotherapy and radiotherapy, and potential future targets for therapeutic intervention.

TGF-β1 down-regulates Th2 development and results in decreased IL-4-induced STAT6 activation and GATA-3 expression

TGF‐β plays an important role in immune regulation in vivo and affects T cell differentiation in vitro. Here we describe how TGF‐β modulates Th2 development in vitro and investigate its mechanisms of action. TGF‐β down‐regulated Th2 development of naive CD4+ Mel‐14high T cells derived from the DO11.10 ovalbumin‐specific TCR‐transgenic mouse, and this was observed both in cultures driven with anti‐CD3 and anti‐CD28 and with splenic APC and antigen. TGF‐β down‐regulated GATA‐3 expression in developing Th2 and these cells showed a diminished IL‐4‐induced STAT6 activation. We found, however, that naive cells driven in Th2 conditions with TGF‐β did not show a significantly decreased STAT6 activation, suggesting that TGF‐β inhibits Th2 development via a STAT6‐independent mechanism.

A Comprehensive Proteomics and Genomics Analysis Reveals Novel Transmembrane Proteins in Human Platelets and Mouse Megakaryocytes Including G6b-B, a Novel Immunoreceptor Tyrosine-based Inhibitory Motif Protein

The platelet surface is poorly characterized due to the low abundance of many membrane proteins and the lack of specialist tools for their investigation. In this study we identified novel human platelet and mouse megakaryocyte membrane proteins using specialist proteomics and genomics approaches. Three separate methods were used to enrich platelet surface proteins prior to identification by liquid chromatography and tandem mass spectrometry: lectin affinity chromatography, biotin/NeutrAvidin affinity chromatography, and free flow electrophoresis. Many known, abundant platelet surface transmembrane proteins and several novel proteins were identified using each receptor enrichment strategy. In total, two or more unique peptides were identified for 46, 68, and 22 surface membrane, intracellular membrane, and membrane proteins of unknown subcellular localization, respectively. The majority of these were single transmembrane proteins. To complement the proteomics studies, we analyzed the transcriptome of a highly purified preparation of mature primary mouse megakaryocytes using serial analysis of gene expression in view of the increasing importance of mutant mouse models in establishing protein function in platelets. This approach identified all of the major classes of platelet transmembrane receptors, including multitransmembrane proteins. Strikingly 17 of the 25 most megakaryocyte-specific genes (relative to 30 other serial analysis of gene expression libraries) were transmembrane proteins, illustrating the unique nature of the megakaryocyte/platelet surface. The list of novel plasma membrane proteins identified using proteomics includes the immunoglobulin superfamily member G6b, which undergoes extensive alternate splicing. Specific antibodies were used to demonstrate expression of the G6b-B isoform, which contains an immunoreceptor tyrosine-based inhibition motif. G6b-B undergoes tyrosine phosphorylation and association with the SH2 domain-containing phosphatase, SHP-1, in stimulated platelets suggesting that it may play a novel role in limiting platelet activation.

Active involvement of Robo1 and Robo4 in filopodia formation and endothelial cell motility mediated via WASP and other actin nucleation-promoting factors

This study aimed to further elucidate the function of Roundabout proteins in endothelium. We show that both Robo1 and Robo4 are present in human umbilical vein endothelial cells (HUVECs) and have knocked expression down using small interfering RNA (siRNA) technology. Roundabout knockout endothelial cells were then studied in a variety of in vitro assays. We also performed a yeast 2‐hybrid analysis using the intracellular domain of Robo4 as bait to identify interacting proteins and downstream signaling. Both Robo1 and Robo4 siRNA knockdown and transfection of Robo4‐green fluorescent protein inhibited endothelial cell movement and disrupted tube formation on Matrigel. Consistent with a role in regulating cell movement, yeast 2‐hybrid and glutathione‐S‐transferase pulldown analyses show Robo4 binding to a Wiskott‐Aldrich syndrome protein (WASP), neural Wiskott‐Aldrich syndrome protein, and WASP‐interacting protein actin‐nucleating complex. We have further shown that Robo1 forms a heterodimeric complex with Robo4, and that transfection of Robo4GFP into HUVECs induces filopodia formation. We finally show using Robo1 knockdown cells that Robo1 is essential for Robo4‐mediated filopodia induction. Our results favor a model whereby Slit2 binding to a Robo1/Robo4 heterodimer activates actin nucleation‐promoting factors to promote endothelial cell migration.—Sheldon, H., Andre, M., Legg, J. A., Heal, P., Herbert, J. M., Sainson, R, Sharma, A. S., Kitajewski, J. K., Heath, V. L., Bicknell, R. Active involvement of Robo1 and Robo4 in filopodia formation and endothelial cell motility mediated via WASP and other actin nucleation‐promoting factors. FASEB J. 23, 513–522 (2009)

A novel method of differential gene expression analysis using multiple cDNA libraries applied to the identification of tumour endothelial genes

BackgroundIn this study, differential gene expression analysis using complementary DNA (cDNA) libraries has been improved. Firstly by the introduction of an accurate method of assigning Expressed Sequence Tags (ESTs) to genes and secondly, by using a novel likelihood ratio statistical scoring of differential gene expression between two pools of cDNA libraries. These methods were applied to the latest available cell line and bulk tissue cDNA libraries in a two-step screen to predict novel tumour endothelial markers. Initially, endothelial cell lines were in silico subtracted from non-endothelial cell lines to identify endothelial genes. Subsequently, a second bulk tumour versus normal tissue subtraction was employed to predict tumour endothelial markers.ResultsFrom an endothelial cDNA library analysis, 431 genes were significantly up regulated in endothelial cells with a False Discovery Rate adjusted q-value of 0.01 or less and 104 of these were expressed only in endothelial cells. Combining the cDNA library data with the latest Serial Analysis of Gene Expression (SAGE) library data derived a complete list of 459 genes preferentially expressed in endothelium. 27 genes were predicted tumour endothelial markers in multiple tissues based on the second bulk tissue screen.ConclusionThis approach represents a significant advance on earlier work in its ability to accurately assign an EST to a gene, statistically measure differential expression between two pools of cDNA libraries and predict putative tumour endothelial markers before entering the laboratory. These methods are of value and available http://www.compbio.ox.ac.uk/data/diffex.html to researchers that are interested in the analysis of transcriptomic data.

Identification of Tspan9 as a novel platelet tetraspanin and the collagen receptor GPVI as a component of tetraspanin microdomains

Platelets are essential for wound healing and inflammatory processes, but can also play a deleterious role by causing heart attack and stroke. Normal platelet activation is dependent on tetraspanins, a superfamily of glycoproteins that function as ‘organisers’ of cell membranes by recruiting other receptors and signalling proteins into tetraspanin-enriched microdomains. However, our understanding of how tetraspanin microdomains regulate platelets is hindered by the fact that only four of the 33 mammalian tetraspanins have been identified in platelets. This is because of a lack of antibodies to most tetraspanins and difficulties in measuring mRNA, due to low levels in this anucleate cell. To identify potentially platelet-expressed tetraspanins, mRNA was measured in their nucleated progenitor cell, the megakaryocyte, using serial analysis of gene expression and DNA microarrays. Amongst 19 tetraspanins identified in megakaryocytes, Tspan9, a previously uncharacterized tetraspanin, was relatively specific to these cells. Through generating the first Tspan9 antibodies, Tspan9 expression was found to be tightly regulated in platelets. The relative levels of CD9, CD151, Tspan9 and CD63 were 100, 14, 6 and 2 respectively. Since CD9 was expressed at 49000 cell surface copies per platelet, this suggested a copy number of 2800 Tspan9 molecules. Finally, Tspan9 was shown to be a component of tetraspanin microdomains that included the collagen receptor GPVI (glycoprotein VI) and integrin α6β1, but not the von Willebrand receptor GPIbα or the integrins αIIbβ3 or α2β1. These findings suggest a role for Tspan9 in regulating platelet function in concert with other platelet tetraspanins and their associated proteins.

The emerging role of tetraspanin microdomains on endothelial cells

Tetraspanins function as organizers of the cell surface by recruiting specific partner proteins into tetraspanin-enriched microdomains, which regulate processes such as cell adhesion, signalling and intracellular trafficking. Endothelial cells appear to express at least 23 of the 33 human tetraspanins, and a number of recent studies have demonstrated their importance in endothelial cell biology. Tetraspanin CD151 is essential for pathological angiogenesis, which may in part be due to regulation of its main partner proteins, the laminin-binding integrins α3β1, α6β1 and α6β4. CD9 and CD151 are essential for leucocyte recruitment during an inflammatory response, through the formation of pre-assembled nano-platforms containing the adhesion molecules ICAM-1 (intercellular adhesion molecule 1) and VCAM-1 (vascular cell adhesion molecule 1), which ultimately coalesce to form docking structures around captured leucocytes. Tetraspanin CD63 also facilitates leucocyte capture by promoting clustering of the adhesion molecule P-selectin. Finally, Tspan12 is required for blood vessel development in the eye, through regulation of Norrin-induced Frizzled-4 signalling, such that Tspan12 mutations can lead to human disease. Future studies on these and other endothelial tetraspanins are likely to provide further novel insights into angiogenesis and inflammation.

ECSM2, An Endothelial Specific Filamin A Binding Protein That Mediates Chemotaxis

Objective—We aimed to characterize the expression and function of a novel transcript that bioinformatics analysis predicted to be endothelial specific, called endothelial-specific molecule-2 (ECSM2). Methods and Results—A full-length cDNA was isolated and predicted ECSM2 to be a putative 205–amino acid transmembrane protein that bears no homology to any known protein. Quantitative polymerase chain reaction analysis in vitro and in situ hybridization analysis in vivo confirmed ECSM2 expression to be exclusively endothelial, and localization to the plasma membrane was shown. Knockdown of ECSM2 expression in human umbilical vein endothelial cells using siRNA resulted in both reduced chemotaxis and impaired tube formation on matrigel, a solubilized basement membrane, both processes involved in angiogenesis. A yeast 2 hybrid analysis using the ECSM2 intracellular domain identified filamin A as an interacting protein. This interaction was confirmed by precipitation of filamin-A from endothelial cell lysates by a GST-tagged intracellular domain of ECSM2. Conclusion—This study is the first to characterize a novel cell surface protein ECSM2 that regulates endothelial chemotaxis and tube formation, and interacts with filamin A. These studies implicate a role for ECSM2 in angiogenesis via modulation of the actin cytoskeleton.

RhoJ/TCL Regulates Endothelial Motility and Tube Formation and Modulates Actomyosin Contractility and Focal Adhesion Numbers

Objective—RhoJ/TCL was identified by our group as an endothelial-expressed Rho GTPase. The aim of this study was to determine its tissue distribution, subcellular localization, and function in endothelial migration and tube formation. Methods and Results—Using in situ hybridization, RhoJ was localized to endothelial cells in a set of normal and cancerous tissues and in the vasculature of mouse embryos; endogenous RhoJ was localized to focal adhesions by immunofluorescence. The proangiogenic factor vascular endothelial growth factor activated RhoJ in endothelial cells. Using either small interfering (si)RNA-mediated knockdown of RhoJ expression or overexpression of constitutively active RhoJ (daRhoJ), RhoJ was found to positively regulate endothelial motility and tubule formation. Downregulating RhoJ expression increased focal adhesions and stress fibers in migrating cells, whereas daRhoJ overexpression resulted in the converse. RhoJ downregulation resulted in increased contraction of a collagen gel and increased phospho–myosin light chain, indicative of increased actomyosin contractility. Pharmacological inhibition of Rho-kinase (which phosphorylates myosin light chain) or nonmuscle myosin II reversed the defective tube formation and migration of RhoJ knockdown cells. Conclusion—RhoJ is endothelial-expressed in vivo, activated by vascular endothelial growth factor, localizes to focal adhesions, regulates endothelial cell migration and tube formation, and modulates actomyosin contractility and focal adhesion numbers.

RhoJ interacts with the GIT–PIX complex and regulates focal adhesion disassembly

ABSTRACT RhoJ is a Rho GTPase expressed in endothelial cells and tumour cells, which regulates cell motility, invasion, endothelial tube formation and focal adhesion numbers. This study aimed to further delineate the molecular function of RhoJ. Using timelapse microscopy RhoJ was found to regulate focal adhesion disassembly; small interfering RNA (siRNA)-mediated knockdown of RhoJ increased focal adhesion disassembly time, whereas expression of an active mutant (daRhoJ) decreased it. Furthermore, daRhoJ co-precipitated with the GIT–PIX complex, a regulator of focal adhesion disassembly. An interaction between daRhoJ and GIT1 was confirmed using yeast two-hybrid experiments, and this depended on the Spa homology domain of GIT1. GIT1, GIT2, &bgr;-PIX (also known as ARHGEF7) and RhoJ all colocalised in focal adhesions and depended on each other for their recruitment to focal adhesions. Functionally, the GIT–PIX complex regulated endothelial tube formation, with knockdown of both GIT1 and GIT2, or &bgr;-PIX phenocopying RhoJ knockdown. RhoJ-knockout mice showed reduced tumour growth and diminished tumour vessel density, identifying a role for RhoJ in mediating tumour angiogenesis. These studies give new insight into the molecular function of RhoJ in regulating cell motility and tumour vessel formation.