Faculty of Biological Sciences
ECBU research1

Endothelial Cell Biology Unit

RESEARCH

Our work currently falls within 4 major project areas:

1) The human LOX-1 scavenger receptor as a pro-inflammatory mediator and regulator of vascular physiology. The LOX-1 scavenger receptor was originally identified as a key endothelial receptor for pro-atherogenic oxidised low-density lipoprotein (OxLDL) in 1997. This integral membrane glycoprotein has an extracellular C-type lectin-like fold and binding to OxLDL stimulates intracellular signalling, gene expression and pro-inflammatory responses including superoxide elevation. Human polymorphisms in LOX-1 alleles are associated with changes in risk for acute coronary syndromes including heart attacks; the mouse knockout model for LOX-1 indicates that this protein contributes to atherosclerotic plaque formation. We are studying how the structure and function of LOX-1 relates to its physiological role in endothelial cells, leukocytes and vascular tissues.

Stephen SL, Freestone K, Dunn S, Twigg M, Homer-Vanniasinkam S, Walker JH, Wheatcroft SB, Ponnambalam S (2010). Scavenger receptors and their potential as therapeutic targets in the treatment of cardiovascular disease. International J Hypertension doi:10.4061/2010/646929.

Thomas JC, Vohra RS, Beer S, Bhatti K, Ponnambalam S, Homer-Vanniasinkam S (2009). Biomarkers in peripheral arterial disease. Trends in Cardiovascular Medicine 19: 147-151.

Vohra RS, Walker JH, Howell GJ, Homer-Vanniasinkam S, Ponnambalam S (2009). The LOX-1 scavenger receptor cytoplasmic domain contains a transplantable endocytic motif. Biochem Biophys Res Commun.  383: 269-274.

Murphy JE, Vohra RS, Dunn S, Holloway Z, Monaco AP, Homer-Vanniasinkam S, Walker JH, Ponnambalam S (2008) Oxidized low-density lipoprotein internalization by the LOX-1 scavenger receptor is dependent on a novel cytoplasmic motif and regulated by dynamin-2. J. Cell Science, 121: 2136-2147.

Dunn S, Vohra RS, Murphy JE, Homer-Vanniasinkam S, Walker JH, Ponnambalam S (2008) The lectin-like oxdized lipoprotein particle scavenger receptor: a pro-inflammatory factor in vascular disease. Biochemical J. 409: 349-355.

Vohra RS, Murphy JE, Walker JH, Homer-Vanniasinkam S, Ponnambalam S (2007) Functional refolding of a recombinant C-type lectin-like domain containing intramolecular disulfide bonds. Protein Expression & Purification. 52: 415-421.

*Murphy JE, Tacon D, Tedbury P, Hadden JM, Knowling S, Sawamura T, Peckham M, Phillips SEV, Walker JH, Ponnambalam S (2006) LOX-1 mediates calcium-dependent recognition of phosphatidylserine and apoptotic cells. Biochemical J 393: 107-115. *Cover article of centenary issue.

Vohra RS, Murphy JE, Walker JH, Ponnambalam S, Homer-Vanniasinkam S (2006) Atherosclerosis and the LOX-1 scavenger receptor. Trends in Cardiovascular Medicine 16: 60-64.

Murphy JE, Tedbury P, Homer-Vanniasinkam S, Walker JH, Ponnambalam S (2005) Biochemistry and cell biology of mammalian scavenger receptors. Atherosclerosis 182: 1-15.

 

2) Vascular endothelial growth factor receptor (VEGFR) function and regulation of endothelial physiology. The VEGF family of growth factors regulate many aspects of vascular physiology including vasculogenesis, angiogenesis, blood pressure control and disease states. Endothelial cells express specific membrane-bound receptors that bind such ligands with high affinity, triggering diverse physiological responses. VEGFR1 and VEGFR2 are endothelial receptor tyrosine kinases that bind to VEGF-A, a major mammalian growth factor that regulates vascular development and homeostasis. The human VEGF-A gene encodes at least 6 different splice variants; VEGF-A-165 binds to both VEGFR1 and VEGFR2 with high picomolar affinity. Our current work is directed at understanding how VEGFR1 and VEGFR2 membrane proteins undergo phosphorylation, ubiquitination, trafficking, signalling and degradation in relation to endothelial prperties such as vasodilation, cell proliferation and sprouting of new blood vessels.

Latham AM, Bruns AF, Kanakala J, Johnson AP, Fishwick CWG, Homer-Vanniasinkam S, Ponnambalam S (2011) Indolinones and anilinophthalazines differentially target VEGF-A and bFGF-mediated responses in primary human endothelial cells. British J Pharmacology In press.

Ponnambalam S (2011) Different sorts for different sprouts. Blood In press.

Jopling HM, Howell GJ, Gamper N, Ponnambalam S (2011) The VEGFR2 receptor tyrosine kinase undergoes constitutive endosome-to-plasma membrane recycling. Biochem. Biophys Res Comm E-pub ahead of print.

Ponnambalam S, Alberghina M (2011) Evolution of a VEGF-regulated vascular network from a neural guidance system. Molecular Neurobiology 43:192-206.

Ulyatt C, Walker JH, Ponnambalam S (2011) Hypoxia differentially regulates VEGFR1 and VEGFR2 levels and alters intracellular signaling and cell migration in endothelial cells. Biochem. Biophys Res Comm 404:774-779.

Ulyatt C, Walker JH, Ponnambalam S (2011) Hypoxia differentially regulates VEGFR1 and VEGFR2 levels and alters intracellular signaling and cell migration in endothelial cells. Biochem. Biophys Res Comm 404: 774-779.

Latham AM, Molina-Paris C, Homer-Vanniasinkam S, Ponnambalam S (2010). An integrative model for vascular endothelial growth factor A as a tumour biomarker. Integrative Biology 2: 397-407.

Bruns AF, Herbert SP, Odell AF, Jopling HM, Hooper NM, Zachary IC, Walker JH, Ponnambalam S (2010) Ligand-stimulated VEGFR2 signaling is regulated by co-ordinated trafficking and proteolysis. Traffic. 11: 161-174.

Bruns AF, Bao L, Walker JH, Ponnambalam S (2009) VEGF-A-stimulated signalling in endothelial cells via a dual receptor tyrosine kinase system is dependent on co-ordinated trafficking and proteolysis . Biochem Soc Trans. 37: 1993-1197.

Ponnambalam S (2009) 'Molecular & Cellular Mechanisms of Angiogenesis' 2009 Biochemical Society focused meeting report and analysis. Target Intelligence Service Report, Current Biodata.

Jopling HM, Odell AF, Hooper NM, Zachary IC, Walker JH, Ponnambalam S (2009) Rab GTPase regulation of VEGFR2 trafficking and signaling in endothelial cells. Arterioscl. Thromb. Vasc. Biol. 29: 1119-1124.

Mittar S, Ulyatt C, Howell GJ, Bruns AF, Zachary I, Walker JH, Ponnambalam S (2009) VEGFR1 receptor tyrosine kinase localization to the Golgi apparatus is calcium-dependent. Exp. Cell Res. 315: 877-889 .

Ewan LC, Jopling HM, Jia H, Mittar S, Bagherzadeh A, Howell GJ, Walker JH, Zachary I, Ponnambalam S (2006) Intrinsic tyrosine kinase activity is required for VEGFR2 ubiquitination, sorting and degradation in endothelial cells. Traffic 7: 1270-1282.

Howell G, Herbert S, Smith, JM, Ewan LC, Mittar S, Mohammed M, Hunter A, Simpson N, Turner A, Zachary I, Walker JH, Ponnambalam S (2004) Endothelial cell confluence regulates Weibel-Palade body formation. Mol Membr Biol 21: 413-421.

 

3) Phospholipase A2 enzymes that regulate vascular physiology. Arachidonic acid (AA) is liberated from cellular phospholipids by the action of phospholipase A2 enzymes (PLA2s). This freely diffusible AA is converted into intermediates that are substrates for cyclo-oxygenase enzymes that produce prostaglandins and leukotrienes that have diverse regulatory effects on cellular proliferation, inflammation and blood pressure. The PLA2 enzymes can be classified into three main groups according to their calcium requirement and enzymatic activities: the secretory PLA2s, the cytosolic PLA2s (cPLA2), and the calcium-independent PLA2s (iPLA2). Different studies indicate that multiple AA mobilization mechanisms under the regulation of distinct PLA2s co-exist in cells to regulate vascular responses. We are studying how cPLA2alpha and iPLA2 association with different membrane systems regulates the production of AA and endothelial cell cycle progression. Such studies have implications for cardiovascular disease (blood vessel repair) and cancer (tumour progression).

Herbert SP, Odell AF, Ponnambalam S, Walker JH (2009) Activation of cytosolic phospholipase A2a as a novel mechanism regulating endothelial cell cycle progression and angiogenesis. J. Biol. Chem. 284: 5784-5796.

Herbert SP, Odell AF, Ponnambalam S, Walker JH (2007) The confluence-dependent interaction of cPLA2a with annexin A1 regulates endothelial cell prostaglandin E2 generation. J. Biol. Chem. 282: 34468-34478.

Herbert SP, Walker JH (2006) Group VIA calcium-independent cytosolic phospholipase A2alpha mediates endothelial S phase progression. J Biol Chem 281: 35709-35716.

Herbert SP, Ponnambalam S, Walker JH (2005) Cytosolic phospholipase A2alpha mediates endothelial cell proliferation and is inactivated by association with the Golgi apparatus Mol Biol Cell 16: 3800-3809.

Grewal S, Herbert SP, Ponnambalam S, Walker JH (2005) Cytosolic phospholipase A2alpha localises to different endothelial membrane compartments in an agonist and calcium-dependent manner. FEBS Journal 272: 1278-1290.

Grewal S, Smith JM, Ponnambalam S, Walker JH (2004) Stimulation-dependent recruitment of cytosolic phospholipase A(2)alpha to EA.hy.926 endothelial cell membranes leads to calcium-independent association. Eur J Biochem 271: 69-77.

Grewal S, Ponnambalam S, Walker JH (2003) Golgi localisation of cytosolic phospholipase A2alpha in the human A549 epithelial cell line. J Cell Sci 116: 2303-2110.

Grewal S, Morrrison EE, Ponnambalam S, Walker JH (2002) Nuclear localisation of cytosolic phospholipase A2alpha in the EA.hy.926 human endothelial cell line is proliferation dependent and modulated by phosphorylation. J Cell Sci 115: 4533-4543.

 

4) Regulation of membrane traffic and signalling in exocytic and endocytic pathways. There is a long-standing interest in how membrane traffic pathways are regulated in all eukaryotes. We are using membrane proteins as probes to understand how vesicle formation, trafficking and signalling is co-ordinated in health and disease states. Key model membrane proteins include the trans-Golgi network (TGN) residents TGN46 and the MNK (ATP7A) copper transporter. A key focus is the formation of vesicles at the trans-Golgi network and the retrieval, recycling and sorting of plasma membrane receptors from the endosome back to the TGN.

Bao L, Redondo C, Findlay JBC, Walker JH, Ponnambalam S (2009) Deciphering soluble and membrane protein function using yeast systems. Mol. Membr. Biol. 26: 137-135.

Netherton CL, McCrossan MC, Denyer M, Ponnambalam S, Armstrong J, Takamatsu HH, Wileman TE (2006) African swine fever virus causes microtubule dependent dispersal of the trans-Golgi network and slows delivery of membrane protein to the plasma membrane. J Virol. 80: 11385-11392.

Howell GJ, Holloway Z, Cobbold C, Monaco AP, Ponnambalam S (2006) Cell biology of membrane trafficking in human disease. Intl Rev Cytol  252: 1-69.

*Cobbold C, Coventry J, Ponnambalam S, Monaco AP (2004) Actin and microtubule regulation of trans-Golgi network architecture, and copper-dependent protein transport to the cell surface. Mol Membr Biol 21: 59-66. *Cover article.

Cobbold C, Coventry J, Ponnambalam S, Monaco AP (2003). The Menkes disease ATPase (ATP7A) is internalized via a Rac1-regulated, clathrin- and caveolae-independent pathway. Hum Mol Genet 12: 1523-1533.

Ponnambalam S, Baldwin SA (2003) Constitutive protein secretion from the trans-Golgi network to the plasma membrane. Mol Membr Biol 20: 129-139.

Cobbold C, Monaco AP, Sivaprasadarao A, Ponnambalam S (2003) Aberrant trafficking of transmembrane proteins in human diseases. Trends Cell Biol 13: 639-647.