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

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TTTTTTTTTT T7 promoter

T7 polymerase

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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