Bio-Asp Road Show
May 19th, 2004
Dr. Ann Pascale BijnensDepartment PathologyUniversity 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 artery Plaque with a thrombus
Advanced plaqueEarly plaqueNormal artery Plaque 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 made cDNA, nylon membrane, hand-made human
• Custom made cDNA, nitrocellulose, Bio-Mek 2000 human
• Clontech Atlas human cDNA 7734-1 human
Atlas mouse cDNA 7741-1
mouse
Micro-arrays
• Incyte UniGEM-V human
Unigene 1 mouse
• Affymetrix HG-U133A human
• Agilent Oligo mouse Development mouse
• Custom-made Oligo, glass slides, spotted human
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
MaastrichtAmsterdam Leiden
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
E S R
E S R
E S R
Laser Capture Microdissection
film
tissue
laserbeam
Laser Capture Microdissection
T7 based amplification
AAAAAAAAAA
TTTTTTTTTT T7 promoter
T7 polymerase
AAAAAAAAAA
AAAAAAAAAA
AAAAAAAAAA
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