Bio-Asp Road Show May 19 th, 2004 Dr. Ann Pascale Bijnens Department Pathology University Maastricht Implementation of Bio-Asp analysis tools in NWO Genomics.

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Presentation transcript:

Bio-Asp Road Show May 19 th, 2004 Dr. Ann Pascale Bijnens Department Pathology University Maastricht Implementation of Bio-Asp analysis tools in NWO Genomics project

What is atherosclerosis? inflammatory disease of medium and large arteries underlying cause of cardiovascular diseases (acute myocardial infaction, stroke, peripheral ischemic vascular disease) thickening and sclerosis of vessel wall progressive disease

Plaque progression throughout atherosclerosis Normal arteryPlaque with a thrombus Advanced plaqueEarly plaqueNormal arteryPlaque with a thrombus Advanced plaqueEarly plaque - endothelial cells - macrophages -smooth muscle cells -necrotic core

Identification and validation of genes differentially expressed during atherosclerosis Identification of key factors in atherosclerosis (e.g. inflammation, matrix turn-over, lipid metabolism) In vitro / in vivo validation Differential gene expression in atherosclerosis

Human samples: unique collection of vascular specimen with various stages of atherosclerosis (MPTC) Mouse samples: various mouse models with atherosclerosis (ApoE-/- mouse, ApoE*3Leiden mouse) Human versus mouse

Bio-informatical platforms in use Macro-arrays Custom madecDNA, nylon membrane, hand-madehuman Custom madecDNA, nitrocellulose, Bio-Mek 2000 human Clontech Atlas human cDNA human Atlas mouse cDNA mouse Micro-arrays IncyteUniGEM-V human Unigene 1 mouse Affymetrix HG-U133Ahuman AgilentOligo mouse Developmentmouse Custom-madeOligo, glass slides, spottedhuman

Future Development of large database with gene profiles of human atherosclerotic plaques that differ in plaque and / or patient characteristics Large-scale gene analysis to determine atherosclerosis - specific upstream key-regulators of atherosclerosis in mouse and man

NWO Genomics program MaastrichtAmsterdamLeiden M. Daemen K. Cleutjens A.P. Bijnens N. Kisters H. Pannekoek A. Horrevoets and co-workers Th. van Berkel J. Kuiper and co-workers

Design NWO genomics study Gene expression in macrophages and endothelial cells Laser capture micro-dissection from whole mount plaques Cell cultures Human and mouse plaques Comparison with expression profiles of macrophages and endothelial cells of non-atherosclerotic tissue Identification of key upstream regulators

Micro array analysis Higher order bio-informatics Human / murine samples Macrophages + Endothelial cells early lesions stable lesions ruptured plaques non-atherosclerotic tissue

Identification of key regulators ESR ESR ESR

Laser Capture Microdissection film tissue laserbeam

Laser Capture Microdissection

T7 based amplification AAAAAAAAAA TTTTTTTTTT T7 promoter T7 polymerase AAAAAAAAAA aRNA RNA

Optimal translation of bio-informatics data to biologically important processes Array data: intensity sample versus intensity reference Statistically relevant differences Which biological pathways are involved?

Implementation of Bio-Asp tools Rosetta Resolver Database for micro-array experiments intensities experimental details details samples Accessible for the participating groups Uniform processing of the micro-array data (error models, Lowess normalisation)

Implementation of Bio-Asp tools Spotfire Analysis array data Various clustering methods Links to programs to elucidate pathways Visualisation

Conclusion Advantages of Bio-Asp Availability of analysis tools that are easy to use accessible at different sites widely spread in the scientific world Training possibilities Relatively low cost