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Group Leader: Levon Khachigian Overview: Professor Khachigian is a molecular biologist with specific interests in transcription factors that control the expression of growth factor and cell cycle regulatory genes in vascular (endothelial and smooth muscle) cells, and in novel strategies exploiting this information in the development of interventional approaches to treat and/or prevent vascular occlusive and other proliferative disorders. Professor Khachigian's research group (comprising basic scientists and cardiologists) at the UNSW Centre for Vascular Research (School of Medical Sciences, Department of Pathology) is focused on five principal areas of research:
Isolation and characterization of new genes induced or repressed by vascular cell injury
Mechanisms of signaling and transcriptional control in vascular endothelial cells and smooth muscle cells
Molecular control of vascular cell proliferation and apoptosis
DNAzymes as molecular and therapeutic tools
Models of neointima formation (SMC hyperplasia), endothelial growth and angiogenesis, vascular leakiness, inflammation, myocardial ischemia-reperfusion injury, and tumour growth |  |
If you’re interested in studying for a PhD in the Khachigian Laboratory in 2007,
please email L.Khachigian@unsw.edu.au asap, sending your CV and undergrad results. A. Recent highlights from the Khachigian Laboratory include:
Conventional anti-inflammatory strategies induce multiple side effects, highlighting the need for novel targeted therapies. We have shown that knockdown of the basic-region leucine zipper protein, c-Jun, by a catalytic DNA molecule, Dz13, suppresses vascular permeability and transendothelial emigration of leukocytes in murine models of vascular permeability, inflammation, acute inflammation and rheumatoid arthritis. Treatment with Dz13 reduced vascular permeability due to cutaneous anaphylactic challenge or VEGF administration in mice. Dz13 also abrogated monocyte-endothelial cell adhesion in vitro and abolished leukocyte rolling, adhesion and extravasation in a rat model of inflammation. Dz13 suppressed neutrophil infiltration in the lungs of mice challenged with endotoxin, a model of acute inflammation. Finally, Dz13 reduced joint swelling, inflammatory cell infiltration and bone erosion in a mouse model of rheumatoid arthritis. Mechanistic studies showed that Dz13 blocks cytokine-inducible endothelial c-Jun, E-selectin, ICAM-1, VCAM-1 and VE-cadherin expression but has no effect on JAM-1, PECAM-1, p-JNK-1 or c-Fos. These findings implicate c-Jun as a useful target for anti-inflammatory therapies.
We have shown that DNAzymes targeting a specific motif in the 5' untranslated region of early growth response (Egr-1) mRNA, inhibit Egr-1 protein expression, microvascular endothelial cell replication and migration, and microtubule network formation on basement membrane matrices. Egr-1 DNAzymes blocked angiogenesis in subcutaneous Matrigel plugs in mice, an observation that was independently confirmed by plug analysis in Egr-1-deficient animals, and inhibited MCF-7 human breast carcinoma growth in nude mice. Egr-1 DNAzymes suppressed tumor growth without influencing body weight, wound healing, blood coagulation or other hematological parameters. These agents inhibited endothelial expression of fibroblast growth factor (FGF)-2, a proangiogenic factor downstream of Egr-1, but not that of vascular endothelial growth factor (VEGF). Egr-1 DNAzymes also repressed neovascularization of rat cornea. Thus, microvascular endothelial cell growth, neovascularization, tumor angiogenesis and tumor growth are processes that are critically dependent on Egr-1.
Percutaneous transluminal coronary angioplasty is a frequently used interventional technique to reopen arteries that have narrowed because of atherosclerosis. Restenosis, or renarrowing of the artery shortly after angioplasty, is a major limitation to the success of the procedure and is due mainly to smooth muscle cell accumulation in the artery wall at the site of balloon injury. We have demonstrated that the anti-angiogenic sulfated oligosaccharide, PI-88, inhibits primary vascular smooth muscle cell proliferation and reduces intimal thickening 14 days after balloon angioplasty of rat and rabbit arteries. PI-88 reduced heparan sulfate content in the injured artery wall and prevented change in smooth muscle phenotype. However, the mechanism of PI-88 inhibition was not merely confined to the antiheparanase activity of this compound. PI-88 blocked extracellular signal-regulated kinase-1/2 (ERK1/2) activity within minutes of smooth muscle cell injury. It facilitated FGF-2 release from uninjured smooth muscle cells in vitro, and super-released FGF-2 after injury while inhibiting ERK1/2 activation. PI-88 inhibited the decrease in levels of FGF-2 protein in the rat artery wall within 8 minutes of injury. PI-88 also blocked injury-inducible ERK phosphorylation, without altering the clotting time in these animals. Optical biosensor studies revealed that PI-88 potently inhibited (Ki 10.3 nmol/L) the interaction of FGF-2 with heparan sulfate. These findings show for the first time the capacity of this sulfated oligosaccharide to directly bind FGF-2, block cellular signaling and proliferation in vitro, and inhibit injury-induced smooth muscle cell hyperplasia in two animal models. As such, this study demonstrates a new role for PI-88 as an inhibitor of intimal thickening after balloon angioplasty.
Platelet-derived growth factor (PDGF) has been implicated in the pathogenesis of vascular occlusive disorders such as atherosclerosis and restenosis in part due to its regulation of smooth muscle cell phenotype. The molecular mechanisms regulating the expression of PDGF-Ralpha, which binds all known dimeric forms of PDGF except PDGF-DD, are poorly understood. We have demonstrated that the winged helix-turn-helix proto-oncogene Ets-1 controls PDGF-Ralpha transcription and mRNA expression in smooth muscle cells. Mutational analysis, electrophoretic mobility shift assay, and chromatin immunoprecipitation revealed the existence of a reverse Ets binding motif (-45TTCC-42) in the proximal region of the PDGF-Ralpha promoter, which bound both recombinant and endogenous Ets-1. Ets-1-inducible PDGF-Ralpha expression depended on the integrity of both the -45TTCC-42 motif and the -61G10(-52) element, which resides upstream of -45TTCC-42 and mediates Sp1 induction. Hydrogen peroxide (H2O2) at nanomolar concentrations stimulated levels of Ets-1 and increased PDGF-Ralpha transcription and mRNA expression without affecting Sp1 expression. H2O2 activation of the PDGF-Ralpha promoter was abolished by disrupting -45TTCC-42 or -61G10(-52). These studies identify a functional Ets motif in the PDGF-Ralpha promoter that plays a pivotal role in agonist-inducible PDGF-Ralpha transcription.
In efforts to better understand mechanisms of repression of PDGF expression, we isolated a nuclear factor that interacts with the proximal region of the PDGF-A promoter using bulk binding assays and chromatography techniques. Peptide mass fingerprint and supershift analysis revealed this DNA-binding protein to be NF1/X. NF1/X repressed PDGF-A promoter-dependent transcription and endogenous mRNA expression, which was reversible by oligonucleotide decoys bearing an NF1/X-binding site. Mutation in the DNA-binding domain of NF1/X abolished its repression of PDGF-A promoter. NF1/X antagonized the activity of a known activator of the PDGF-A chain, Sp1, by inhibiting its occupancy of the proximal PDGF-A promoter. NF1/X physically and specifically interacts with Sp1 via its subtype-specific domain and blocks Sp1 induction of the promoter. NF1/X residues 311-416 mediated NF1/X suppression of basal PDGF-A transcription, whereas residues 243-416 were required for NF1/X repression of Sp1-inducible promoter activity. These findings demonstrate that repression of PDGF-A gene transcription is governed by interplay between NF1/X and Sp1.
PDGF-D is the newest member of the PDGF family. The molecular mechanisms regulating its transcription are not known. Using primer extension analysis, we mapped a single transcriptional start site to the ccAGCGC motif with several potential Ets motifs located upstream. Ets-1, but not Ets-1 bearing only the DNA-binding domain, activates the PDGF-D promoter and mRNA expression in SMCs. Ets site D3 ((-470)GGAT(-467)) is singly required for basal and Ets-1-inducible PDGF-D promoter-dependent expression. D3 supports the interaction of endogenous and recombinant Ets-1 and Sp1. Sp1, like Ets-1, induces PDGF-D transcription and mRNA expression, which is blocked by mutant Ets-1. H2O2 stimulates Ets-1, but not Sp1, and activates D3-dependent PDGF-D transcription. Ets-1 and Sp1 siRNA block peroxide-inducible PDGF-D expression. Angiotensin II (ATII) induction of PDGF-D and Ets-1 was blocked by prior incubation of the cells with PEG-catalase, but not BSA, indicating that ATII-inducible Ets-1 and PDGF-D expression is mediated via H2O2. Thus, 2 separate trans-acting factors regulate PDGF-D transcription, alone and in response to oxidative stress. B. Key Publications
Please click on http://notes.med.unsw.edu.au/resinterests.nsf/sw/8571226 C. Group Members: D. Funding sources
NHMRC Program
ARC Discovery
Cancer Council
Diabetes Australia
National Heart Foundation of Australia Grant-in-Aid
E. Collaborations
Professor Khachigian has numerous active scientific collaborations locally and internationally.
F. Patent and Commercial Activities
Levon Khachigian has a strong record of translational research that has involved commercial funding of in-laboratory R&D by numerous local international pharmaceutical/biotech companies. This is built on a suite of patents. He is currently developing "molecular assassin" anti-gene technology which has therapeutic implications to common problems such as eye disease, arthritis, myocardial ischemia-reperfusion injury, restenosis, and cancer. NewSouth Innovations (the commercialisation arm of UNSW) is currently in negotiations with commercial organisations on the development of novel gene-therapeutic and “anti-gene”-therapeutic applications in vascular diseases and cancer.
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