Professor Moshe Wolman:

Pioneer in Histochemistry
Macrophages and Atherosclerosis
Histopathology of
atherosclerosis
Raymond Coleman, Tony Hayek,
Shlomo Keidar & Michael Aviram

Rappaport Faculty of Medicine

Haifa, Israel
Cardiovascular disease: the major
cause of premature mortality
Coronary blockage
 Risk factors for atherosclerosis
• Genetic (inherited)
• Gender (maleness)
• Intimal injury
• Vascular hemodynamics (shear stress)
• Hypertension
• Inflammation
• Infection (Chlamydia) and immune responses (?)
• Lipid disorders (hypercholesterolemia)
• Diabetes
• Cigarette smoking
• Elevated homocysteine
• Elevated iron
• Elevated C reactive protein
• Lifestyle (sedentary, eating disorders, Western diet)
• Cardiovascular aging changes
 Risk factors?
More than you imagine
Hypercholesterolemia is widely regarded
as a major risk factor for atherosclerosis
Clinton’s diet
 Apolipoprotein E-deficient mice
• Since the early 1990s the apolipoprotein E-
deficient mouse has provided a useful animal
model of hypercholesterolemia and the
progressive development of atherosclerotic
plaque similar to that of humans
• The earliest stages of plaque development are
seen within 6-8 weeks
• Quantitative image analysis of plaque permits
research on the effectiveness of drugs or food
additives in retarding plaque development
The apolipoprotein E-deficient mouse
develops spontaneous atherosclerotic
plaque
Sites of atherosclerotic plaque in
apolipoprotein E-deficient mice

Nakashima et al
Arterioscler Thromb
)14: 133 )1994
 Aortic arch and atherosclerotic plaque

• The aortic arch is a major site for the

development of atherosclerotic plaque
• Progressive changes in plaque
development were followed for up to 17
months in apoE-deficient mice
• Epon-embedded sections were stained
with alkaline toluidine blue
Dissected aortic arch
Left Right

mm
 Aortic arch sampling

Ni et al
J Nutr 128:1884-9
))1994
Aortic arch sampling
Macrophages have complex
roles in developing plaque

)Hajjar & Nicholson Am Sci 83:459-467 )1995

 Osmium-stained lipid in
macrophages inTunica intima
Macrophages inT.intima
Macrophages inT.intima
Macrophages form lipid pools
?Macrophage escape
Macrophages
Ultrastructure of intimal
macrophage
Early fatty streak
Fatty streak
Early fatty streak
Advanced fatty streak
Advanced fatty streak
Elastic membranes ofT.media rupture
Smooth muscle changes in the T. media
Smooth muscle changes in theT. media
Invasion of theT.adventitia
Lipid pools and cholesterol
Lipid pools can be large
Fibrous cap and lipid pools
Cholesterol
Cholesterol
Cholesterol and lipid pools
Cholesterol
Cholesterol
Cholesterol
Cholesterol
Precalcification
Cartilage-like cells and precalcification
Early calcification
Calcification
Calcification
Occlusion of the lumen
Occlusion of the lumen
Occlusion of the lumen
Occlusion of the lumen
Occlusion of the lumen
Occlusion of the lumen
Occlusion of the lumen
Plaque lesion size in aortic arch of apolipoprotein E-
deficient mice according to age
Cumulative data from our studies over a 10 year period

350000
300000
square micrometers

250000
200000

150000
100000

50000
0
6 8 10 12 14 16 20 24 32 44
weeks
Plaque progression in apoE-
deficient mice
)Meir KS, Leitersdorf E ATVB 24:1007 )2004
 Plaque development

)Navab et al Am J Cardiol 76:18C-23C )1995

Monocytes adhere to endothelium as precursors of
intimal macrophages
 Plaque development

)Navab et al Am J Cardiol 76:18C-23C )1995

 Plaque development

)Hajjar & Nicholson Am Sci 83: 459-467 )1995

 Plaque development:
hemodynamic forces, monocytes
and adhesion molecules

)Hajjar & Nicholson Am Sci 83: 459-467 )1995

Complex roles of macrophages

)Hajjar & Nicholson Am Sci 83: 459-467 )1995

LDL oxidation and antioxidants

)Schwerke DC J Nutr Biochem 9: 424-445 )1998

Cholesterol synthesis
 iNOS
Immunohistochemical localization in aortic arch

6 WEEKS 8 WEEKS

10 WEEKS 12 WEEKS
 VEGF
Immunohistochemical localization in aortic arch

6 WEEKS 8 WEEKS

10 WEEKS 12 WEEKS
 MMP 2
Immunohistochemical localization in aortic arch

6 WEEKS 8 WEEKS

10 WEEKS 12 WEEKS
iNOS
The iNOS expression increases over time, suggesting that iNOS
accelerates lesion progression from an early time point and that the
.amount of iNOS-mediated injury increases over time

VEGF
VEGF immunostaining shows a time-dependent increase in VEGF
expression; in early steps )6-8 weeks). The positivity is present
mainly in theTunica intima layer near the injury site. At 10-12 weeks
we observe a spread of immunostaining also in theTunica media
.layer

MMP2
MMP2 immunostaining shows a similar pattern of MMP2 expression at
.all times studied
 The problem of unstable plaque
• Most heart attacks occur when an area of
unstable plaque ruptures and breaks away
• This causes a blood clot and subsequent
abrupt flow blockage
• This may occur in areas of arteries where
plaque was not obstructing the artery
• Unstable plaque is far more dangerous
than stable )fibrous cap) plaque
The problem of plaque
Unstable plaque
Stable plaque
Unstable plaque
Unstable plaque
Unstable plaque
Unstable plaque rupture
Tissue factor in thrombus formation
)ATVB 24: )2004
Plaque rupture
 Plaque rupture

)Navab et al. Am J Cardiol 76: 18C-23C )1995

 Plaque rupture

)Hajjar & Nicholson Am Sci 83: 459-467 )1995

Unstable plaque
 ?How to reduce risk of plaque rupture

Increase stability by: Reduce risk factors by:

• Decreasing lipid core • Decreasing
• Decreasing vascular mechanical stress
wall inflammation • Decreasing blood
• Strengthening the pressure
fibrous cap • Decreasing cardiac
rate
Mindguard “diverter” for
unstable plaque
 Lipoproteins:
the good, the bad and the ugly
Damage control
 Plaque retardation?
Exercise, healthy diet, cholesterol-reducing drugs
?Jogging
?Statins for everybody
Damage control
Functional Imaging of Plaque
)RP Choudhury et al. Nature Reviews 3:913-925 )2004
Coronary arteries in apolipoprotein
E-deficient mice
• Apolipoprotein E-deficient mice with
extensive atherosclerotic plaques in the
aorta, surprisingly, do not show any
pathological changes in their coronary
arteries
• Lack of histopathological changes in
coronary arteries in hypercholesterolemic
mice means these are not a suitable
model to study coronary disease
 Coronary arteries in hypercholesteremic
apolipoprotein E-deficient mice remain plaque-free
 Conclusions
• Animals, unlike humans, do not suffer
from heart attacks (infarcts) and rarely
develop spontaneous atherosclerosis
• The apolipoprotein E-deficient mouse
model has proved to be the best and
most rewarding animal research model
for the study of hypercholesterolemia,
atherosclerosis and to test the efficacy
of drug intervention and therapy
Take home message

”Prevention is better than cure“

Take home message

”An apple a day keeps the doctor away“

Old English proverb