Plaque temperature heterogeneity is associated with

macrophage accumulation and metalloproteinase activity

R. Krams, LCA van Damme

Dept. Experimental Cardiology, Thoraxcenter, Rotterdam, The Netherlands

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

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.

MethodsIn 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

start

end

3F Fogarty

50 mm

Infra renalaorta

Renal artery

Denudation area

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

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

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

Analysis III• MMP activity in a zymogram

-SDS PAGE gel electroforese

-separation on molecular weight

-substrate: gelatin

ProtocolInducing atherosclerosis

Perform thermography after 2 months

Hot Cold

Isolate area Isolate area

MMP-activity / cel distribution MMP-activity / cel distribution

Compare

Distance (mm) from renal artery

0 5 10 15 20 25 30 35 40 45 50 55

T (°

C) f

rom

refe

renc

e

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

T (°

C) f

rom

refe

renc

e

-0.2

-0.1

0.0

0.1

length (mm) vs Mean without blood flowlength (mm) vs Mean with blood flow

Results IThermograph (with and without blood flow)

Hot spot 1

Hot spot 2Cold area

Example 1

Length from renal artery (mm)

0 5 10 15 20 25 30 35 40 45 50 55

T (°

C) f

rom

refe

renc

e ar

ea

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

T (°

C) f

rom

refe

renc

e ar

ea

-0.01

0.00

0.01

0.02

0.03

length (mm) vs Mean without blood flowlength (mm) vs Mean with blood flow

Example 2

Results IThermograph (with and without blood flow)

T (°C) from reference

0.1 0.2 0.3 0.4 0.5 0.6 0.7

VI-i

ndex

0

1

2

3

VI-indexRegression between VI-index and T R=0.8, p=0.013

Results II

Results II

T (°C) from reference

0.1 0.2 0.3 0.4 0.5 0.6 0.7

Mø

(%) i

n th

e pl

aque

0

5

10

15

20

25

30

møRegression between mø(%) and T (°C), R = 0.7 p = 0.020

Results IIIMMP-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 artery0 10 20 30 40 50 60

T (°

C) f

rom

refe

renc

e

0.00

0.25

0.50

0.75

MMP2activeproMMP2 MMP9

MM

P a

ctiv

ity (p

ixel

s/m

m2 )

0

5000

10000

Hot regionsReference rgeions

*

*

* p<0.05

Results III

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

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

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

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

Co-operation

• Catherization laboratoryProf. Dr. PW SerruysDr. W. van der Giessen

• Experimental Cardiology Rotterdam L.C.A van Damme

C. van PeltB. Mousavi GourabiW. MaatD. SegersC. ChengDr. R. KramsDr. D.J. Duncker

• Experimental Cardiology UtrechtDr. G. PasterkampC. SnijderA. Schoneveld

• Thermocore Medical SystemsDr. G. Van LangenhoveDr. T. FlintDr. Y. Yianni

• Middelheim Hospital AntwerpenS. Verheye

M. KockxM. Knaapen