metal ions in heart disease michael j. davies the heart research institute 145 missenden road,...

METAL IONS IN HEART DISEASE

Michael J. Davies

The Heart Research Institute

145 Missenden Road, Camperdown, Sydney, Australia

The Heart Research Institute• Independent Research Institute • Opened in 1989• Bicentennial initiative of

– National Heart Foundation– Cardiologists of Royal Prince Alfred Hospital– NSW State Government

• Non-profit, registered charity• Affiliated to

– Royal Prince Alfred Hospital– University of Sydney

HRI Research Groups

• Cell Biology (Ken Rodgers)

• Clinical Research (David Celermajer / Len Kritharides)

• Endothelial Cell Biology (Philip Barter)

• Free Radical (Mike Davies)

• Lipid Research (Kerry-Anne Rye)

• Molecular Biology (Alison Death)

• Vascular Immunology (Annemarie Hennessy)

Mission statement

Develop new scientific knowledge which will lead to prevention, early detection and reversal of atherosclerosis (the cause of cardiovascular disease).

What is atherosclerosis?• A process that clogs arteries with deposits of

cholesterol and lipids.

What is atherosclerosis?• A process that clogs arteries with deposits of

cholesterol and lipids.

• This reduces the blood supply to the organ that the artery supplies, which can result in serious organ damage.

• Rupture of lesions can release particulate matter that blocks other blood vessels

Consequences of atherosclerosis

• Angina

• Heart attack (myocardial infarction)

• Stroke

• Intermittent claudication

• Gangrene

The cost of atherosclerosis• Major health problem and cost to society

• 1 person dies every 10 minutes• 41% of all deaths arise as a result of atherosclerosis• Coronary heart disease is largest single cause of death

(more than 29,000/year)• Stroke second greatest killer (more than 12,000/year)• Atherosclerosis is a leading cause of long term disability

in adults• $3.7 billion a year (12% of health budget)

Areas of expertise within HRI

• Lipid metabolism• Free radical chemistry and biochemistry• Stem cells• Cell signalling• Protein modification, metabolism and turnover• Cytokines• Endothelial cell function• Diabetes

Specialised equipment / facilities

• Cell isolation and culture (primary and cell lines)

• Isolation and handling of lipoproteins (LDL / HDL)

• Enzyme kinetics and assays

• Quantification of DNA oxidation (HPLC, agarose gels)

• Quantification of normal and oxidised lipids and cholesterol

• Quantification of normal and oxidised / modified proteins (HPLC, 1D and 2D gel electrophoresis, proteomics)

• Quantification of antioxidants (HPLC, GC/MS)

• Radioactive tracer work

• Identification and quantification of radical formation (EPR, NO / O2 /H2O2 electrodes)

Quantification of oxidised DNA bases

X.H2O

8-oxodG2’-deoxyguanosine

Presence of 8-oxodG disrupts base-pairing and gives G-C to A-T transversions

0

200

400

600

800

1000

1200

1400

Non-irradiated 1000 Gy 1500 Gy 2000 Gy Reduced

No added metal ions

+ Cu+

8-o

xo

dG

/ 1

05

par

ent

2'-d

G

(fro

m c

alf

thym

us

DN

A h

ydro

lysa

tes)

Histone H1

* o

* o

*

*

O

H

O

O

H

N

N

N

N

H

O

N

H

2

O

H

O

O

H

N

N

N

N

H

O

N

H

2

O

H

O

O

H

N

N

N

N

H

O

N

H

2

O

H

+.

Quantification of protein oxidation: measurement ofspecific side-chain oxidation products

Tyrosine DOPA

3-chlorotyrosine

3-nitrotyrosine

di-tyrosine

Phenylalanine o-, m-tyrosine

dimers

Tryptophan N-formylkynurenine,

kynurenine,

5-hydroxytryptophan,

7-hydroxytryptophan

Histidine 2-oxo-histidine

Glutamic acid hydroperoxides

Leucine hydroperoxides,

alcohols,

-ketoisocaproic acid,

isovaleric acid,

isovaleraldehyde,

isovaleraldehyde oxime,

carbonyl compounds.

Valine hydroperoxides,

alcohols,

carbonyl compounds.

Lysine hydroperoxides,

alcohols,

carbonyl compounds.

Proline hydroperoxides,

alcohols,

5-hydroxy-2-aminovaleric acid,

carbonyl compounds.

Arginine 5-hydroxy-2-aminovaleric acid

Isoleucine hydroperoxides,

alcohols,

carbonyl compounds

Glycine Aminomalonic acid

Methionine Methionine sulphoxide

Cysteine Cystine,

Oxy acids

Quantification of oxidised proteins in tissue samples

Proteins extracted from tissue samples (normal and diseased),delipidated, and hydrolysed to free amino acids.

HPLC with UV, fluorescence and multi-channel electrochemical detection used to quantify both oxidised species and parent amino acids.

Results expressed as ratio of oxidised species to parent amino acid.

Radiometric and chemiluminescence detectors also available.

Oxidised proteins are present in diseased human arteries

Proteins extracted from human artery samples (normal and advanced carotid lesions) delipidated, and hydrolysed to free amino acids, and analysed by HPLC. Results expressed as moles oxidised species / mole parent.Elevated levels of oxidised lipids and decreased antioxidant concentrations also detected.

0

400

800

1200

DOPA m-Tyr o-Tyr

Normal ArteryCarotid Plaque

0

100

200

300

400

di-Tyr Val.OH1 Leu.OH2

Fu et al, Biochem. J., 1998, 333, 519-525

Oxidation as a cause of atherosclerosis and its complications

• Oxidative events postulated to be important both in the initiation of atherosclerosis and in plaque rupture.

• Some evidence to support this hypothesis, but also considerable evidence for other factors being as, or more, important.

• Potential catalysts of oxidative damage:– Enzyme reactions (lipoxygenase, peroxidases, oxidases, heme

protein reactions)

– Activated inflammatory cells

– Nitric oxide / peroxynitrite

– Trace redox-active metal ions

Oxidative modification model of atherosclerosis

LDL

LDL (bad cholesterol)

Vessel wall

Blood vesselWhite blood cells

LDL

LDL

LDLLDL

LDL

LDL

LDL

Endothelial cells

Oxidative modification of LDL particles

Are trace metal ions present at elevated levels in lesions ?

Oxidants Protein oxidation

Lipid peroxidation

Advanced Human Plaque

Healthy Human Intima

Iron signalOrganic radicals

Magnetic field (G)

Non-destructive technique of EPR spectroscopy used to examine presence of organic radicals and metal ions, particularly Fe(III) and Cu(II) in advanced human lesions.

EPR analysis of human lesions

Correlation of iron levels detected by EPR and ICPMS with tissue status

Plaque Healthy Intima

0

1

2

SamplePlaque Healthy IntimaSmooth M

0

1

2

3

Sample

Mean iron values detected by EPR in lesions significantly higher than that detected in healthy intima samples.

Mean iron values detected by ICPMS in lesions significantly higher than that detected in healthy intima samples.

Comparison of iron levels detected by EPR and ICPMS

0.0 0.5 1.0 1.5 2.0

0.0

0.5

1.0

nmol Fe(III)/mg tissue

Good correlation between the two measurements with EPR measuring ca. 70% of the total iron present

Elevated levels of Cu detected in diseased intima samples compared to normal intima samples.

Cu(II) signals were not detected by EPR.

Plaque Healthy Intima Smooth M

0 .0 0

0 .0 1

0 .0 2

0 .0 3

0 .0 4

S am p le

ICPMS detection of copper in atherosclerotic lesions

Correlation of metal ion levels with other parameters

• No correlation of metal ion levels with:

– Age of tissue donor

– Gender

– Protein levels in lesion

– Calcium levels in lesion

Correlation of iron levels with cholesterol accumulation

EPR-detectable Fe(III) shows a positive correlation with cholesterol levels in lesions.

Is iron accumulation a cause or a consequence of cholesterol accumulation and hence lesion development?

Presence of elevated levels of non-heme, non-ferritin iron consistent with presence of elevated levels of protein and lipid oxidation products and depletion of antioxidants.

0 50 100 150 200 250

0

1000

2000

3000

4000

5000

nmol Fe(III)/mg protein

Unanswered questions

• What are these Fe and Cu complexes ?• Where are they located :- intra- or extra-cellularly ?• Where do they come from ?

– Heme protein degradation ?possible role of oxidative damage or heme oxygenase

– Micro hemorrhage into plaques ?– Smooth muscle cell death ?

• Is it possible to modulate metal ion levels in the artery wall in vivo ?

• Does modulation of metal ion levels affect disease initiation, progression or plaque rupture

• Is it all a secondary artifactual event ?

metal ions in heart disease michael j. davies the heart research institute 145 missenden road,...

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