SHAPE Society

1. Plaque temperature heterogeneity is
associated with
macrophage accumulation and
metalloproteinase activity
R. Krams, LCA van Damme
Dept. Experimental Cardiology, Thoraxcenter,
Rotterdam, The Netherlands

2. Background
• Plaque rupture has been related to a high MMP
activity and macrophage (Mø) accumulation
Galis et al, J. Clin. Invest. 1994
• Recently, regional temperature heterogeneity has
been observed in atherosclerotic plaques in vivo
Verheye et al, Circulation 2002

3. Aim of this study
• To test the hypothesis that temperature
heterogeneity is associated not only with
macrophage accumulation but also with
MMP-activity in atherosclerotic plaques
in vivo.

4. Methods
In 6 NZW rabbits:
• Endothelium was removed (denudation) of
the infra renal aorta
• 2 months 2% cholesterol diet
• Temperature measurement after 2 months of follow up
• Immunohistochemistry for the detection of mø (RAM11, Dako),
smooth muscle cells (1A4), histology for collagen amount
(picro-serious red) and lipid contents (oil red o) and MMP activity
in a zymogram

5. start
end
3F Fogarty
50 mm
Infra renal
aorta
Renal artery
Denudation area

6. Thermography
• Intravascular thermography
• Catheter: Thermocore Medical Systems
• Accuracy: ± 0.01 °C
• Pullback speed: 0.2 mm/sec
• Measurement of temperature difference from a
reference area

7. Analysis I
•Thermography with / without blood flow
-pull back over 6 cm
-mean of 4 thermistors
-mean of 2 pull backs
-reference area set at 0 °C

8. Analysis II
• Histological analysis:
-Mø % of plaque area
-Lipid % of plaque area
-Collagen % of plaque area
-Smooth Muscle Cell(SMC) %
of plaque area
• Vulnerability Index (VI-index):
VI = Mø% + Lipid%
Collagen% + SMC%
Shiomi et al, Atherosclerosis 2001

9. Analysis III
• MMP activity in a zymogram
-SDS PAGE gel electroforese
-separation on molecular weight
-substrate: gelatin

10. Protocol
Inducing atherosclerosis
Perform thermography after 2 months
Hot Cold
Isolate area Isolate area
MMP-activity / cel distribution MMP-activity / cel distribution
Compare

11. Distance (mm) from renal artery
0 5 10 15 20 25 30 35 40 45 50 55
∆T(°C)fromreference
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
∆T(°C)fromreference
-0.2
-0.1
0.0
0.1
length (mm) vs Mean without blood flow
length (mm) vs Mean with blood flow
Results I
Thermograph (with and without blood flow)
Hot spot 1
Hot spot 2
Cold area
Example 1

12. Length from renal artery (mm)
0 5 10 15 20 25 30 35 40 45 50 55
∆T(°C)fromreferencearea
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
∆T(°C)fromreferencearea
-0.01
0.00
0.01
0.02
0.03
length (mm) vs Mean without blood flow
length (mm) vs Mean with blood flow
Example 2
Results I
Thermograph (with and without blood flow)

13. ∆T (°C) from reference
0.1 0.2 0.3 0.4 0.5 0.6 0.7
VI-index
0
1
2
3
VI-index
Regression between VI-index and ∆T R=0.8, p=0.013
Results II

14. Results II
∆T (°C) from reference
0.1 0.2 0.3 0.4 0.5 0.6 0.7
Mø(%)intheplaque
0
5
10
15
20
25
30
mø
Regression between mø(%) and ∆T (°C), R = 0.7 p = 0.020

15. Results III
MMP-9
Pro-MMP-2
MMP-2 active
MMP-9
Pro-MMP-2
MMP-2 active
MMP-9
Pro-MMP-2
MMP-2 active
Length (mm) from renal artery
0 10 20 30 40 50 60
∆T(°C)fromreference
0.00
0.25
0.50
0.75

16. MMP2activeproMMP2 MMP9
MMPactivity(pixels/mm2)
0
5000
10000
Hot regions
Reference rgeions
*
*
* p<0.05
Results III

17. Summary
• We found that temperature has a positive
correlation between mø% and VI-index
• We found more pro-MMP2 and active MMP2
inside the hot area’s compared to the cold area’s
•We found almost no rise in temperature in
presence of blood flow

18. Conclusion I
• Temperature heterogeneity is associated with
a VI-index
• The underlying mechanism of this association
is mø accumulation
• The high MMP-activity suggests that active mø are
detected by temperature changes

19. Conclusion II
• We have seen in our model that it is almost
impossible to measure temperature differences
in presence of blood flow

20. Discussion
• Blood has a cooling effect on the vessel wall, and
blood flow is high in the rabbit’s abdominal aorta
(twice human coronary flow).
• Therefore to exclude a dominant flow effect on our
measurements, we stopped the flow with a balloon

21. Co-operation
• Catherization laboratory
Prof. Dr. PW Serruys
Dr. W. van der Giessen
• Experimental Cardiology Rotterdam
L.C.A van Damme
C. van Pelt
B. Mousavi Gourabi
W. Maat
D. Segers
C. Cheng
Dr. R. Krams
Dr. D.J. Duncker
• Experimental Cardiology Utrecht
Dr. G. Pasterkamp
C. Snijder
A. Schoneveld
• Thermocore Medical Systems
Dr. G. Van Langenhove
Dr. T. Flint
Dr. Y. Yianni
• Middelheim Hospital Antwerpen
S. Verheye
M. Kockx
M. Knaapen