1) Several studies have traced the recruitment of monocytes into atherosclerotic plaques using labeling techniques. Gerrity et al labeled monocytes with FITC and found them adhered to thickened arterial sites. 2) Patel et al labeled macrophages with fluorescent microspheres and found them adhering to plaques in mice. Antibodies against ICAM-1 and integrin reduced monocyte homing. 3) Steinberg et al used a genetic marker to label donor monocytes, finding over 600 donor cells per million in early fatty streaks and over 3,800 in more advanced plaques.

1. Monocyte Recruitment into
Atherosclerotic plaques
Quantification Methods

2. Review of the studies done by:
• Gerrity et al
• Steinberg et al
• Willerson et al
• The potential role of SPIO

3. Initial observation, with the help of EM
(electron microscopy) by Gerrity et all,
led to the hypothesis of “macrophage assisted
lipid clearance mechanism”.
Published in Artery in 1979, he used the swine model
Of hypercholesterolemia. He fed Yorkshire pigs with
high cholesterol diet and sacrifice them at the ages of
12, 15 and 30 weeks. He also used an age-matched
group of pigs as control.
Samples of aortic arch from both lesion and non-lesion
Areas were examined by electron microscopy.

4. Fatty streak lesions develops as early as 15 weeks of
Age. These lesion although becoming more extensive
In area, will not progress beyond fatty streak up to 30
Weeks of age.
EM studies indicated movement of foam cells through
Endothelium overlying the fatty streak lesion into the
Aortic lumen.

5. The above mentioned phenomenon may explain
the stability of fatty streaks for a period of time.
It also explains that macrophages may initially
serve as carriers of lipid material out of the plaque.
However, focal endothelial damage would result
from this process, which may contribute to later
lesion progress by introducing SMC proliferating
factors into the wall.

6. In a 2nd
study by Gerrity et al, published in Atherosclerosis
1988, additional method to trace macrophages was used:
-Monocytes were isolated from peripheral blood of pigs with
Hypercholestrolemia.
-Monocytes were labeled with FITC (fluorocin
Isothiocyanate 1-hydrochloride)
-Labeled monocytes were reinjected back to the animal
-Labeled cells were found in the blood.
-Animals were killed after 9 days, and histology exam was
performed

7. Result
1)FITC-labeled monocytes were found adherent
to the thickened site of the intima but not to
normal areas.
2)Labelled cells are also found in within the
atherosclerotic lesions.

8. In the study by S. Patel, James T. Willerson and
Edward Yeh, published in 1997, peritoneal
macrophages of mouse were labeled with fluorescent
latex microspheres and injected into the blood.
Animals were sacrificed after 48 hours, and tissues
obtained for histology.(ApoE deficient mice)
A quantitative survey was done to detect the number of
labeled macrophages in the arterial wall.
Antibodies to ICAM-1, integrin and E-selectin were
Injected 6-8 hours before macrophage injection.

9. Figure 2. Schematic diagram of the study
area depicting a labeled macrophage
adhering to the plaque.

10. Figure : a, Distribution of macrophages in
the 10-µm section of aorta in a representative
experiment; b, inhibition of macrophage
recruitment by antibody against 4
or
ICAM-1 but not by anti–E-selectin antibody.
The group of mice that were not treated with
antibody is labeled as positive control.
Appropriate isotype-matched antibody was
used for each specific antibody. Mean of each
treatment group is indicated by a bar.
Comparison of the seven treatment groups
was performed with one-way ANOVA
followed by Scheffé's test for post hoc
pairwise comaprisions. All analyses were
done with SAS statistical programs; P<.05
was considered statistically significant.

11. Figure:
Macrophages labeled with
fluorescent microspheres adhere
to atherosclerotic plaques.

12. Results
-Study was performed in 2 groups,(control and test) with
respect to the use of antibodies.
-After 48 hours macrophages were observed adhering to
all stages of plaques.
-The mean number of macrophages in the proximal 1mm
of aortic root was estimated to be 143+17 per aortic root
-Antibodies against ICAM-1 and integrin significantly
reduced the number of macrophage homing.

13. Lewis et al used labeled leukocytes in vivo, by giving
multiple infusions of tritiated thymidine to pigeons
while being repeatedly bled to induce rapid leukocyte
formation.
A harvested tritium-labeled mononuclear cells then
was transfused to a bird with atherosclerosis.
Autoradiography then showed that 2/3 of the
radiographic grains are associated with foam cells,but
the rest was associated with smooth muscle cells and
endothelial cells. (low sensitivity of the test)

14. Steinberg et al, in 2000 published their study regarding
a new method of detecting monocytes in the plaque.
The basic idea is the introduction into a recipient animal
of leukocytes differing from those of the recipient by
virtue of one easily identified and quantified genetic
marker.

15. To minimize manipulation of cells as much as possible
(to avoid activation or damage), Steinberg and his
group used a naturally occurring mutation in the
preliminary experiments, then they transferred the
cells from donor to recipient with a minimum handling.
Rabbit mutation,(NAT-R), loss of gene coding for major
form of arylamine N-acetyl transferase.
10% of New Zealand rabbits are homozygous for a
major deletion in the gene coding for NAT-R.
Homologous deletion inbred were used as donor
and wild type were used as recipient of leukocytes.

16. Methods:
PCR was the tool to detect the mutated leukocyte.
-Due to its extreme sensitivity, this test is able to detect
A band in a dilution of 5 cells in 1 million unmarked
Cells.
-Animals were sacrificed after 65 hours.
-Time course of the disappearance of donor cells from
the blood stream of the recipient animals were
obtained.
-Tissue samples were tested for the presence of donor cell.

17. Figure:
A: Time course of the
disappearance of donor monocytes
purified from the blood of a wild-
type donor(NAT-R) and injected
intravenously into a mutant(NAT-S)
recipient.
B: Time course of the disappearance
of donor monocytes from the blood
of a mutant recipient (NAT-S) after
intravenous injection of 45 ml of
Whole blood from a wild type donor
(NAT-R).

18. Figure : Recovery of donor cells in recipient
tissues 65 hr after injection of purified
NAT-R positive monocytes into an NAT-S
rabbit. The relative quantities of PCR
products are expressed as cpm per µg of
DNA from each tissue (except in the case
of spleen, where the data represent cpm per
20 ng of DNA). Each open circle represents
a value for a sample of DNA carried
separately through PCR and hybridization.

19. Figure: Leukocyte uptake into the aortic lesions of a mutant cholesterol-fed rabbit
(18 wk) 70 hr after transfusion of 45 ml of whole blood from a wild-type donor.
Data represent the amount of 32
P-labeled probe hybridizing with PCR products
generated from 1 µg of aortic DNA. , Values for duplicate DNA samples carried
separately through PCR and hybridization.As indicated in the inset, the successive
thoracic segments represented equal-length samples along the aorta.

20. For the atherosclerotic plaque 2 different setting were
Selected, Fatty streaks and more advanced lesions.
They concluded that 623 per million cells in the early
Fatty streaks were donor leukocytes.
In more advanced stage, the aortic arch showed a
maximum number of 3860 donor leukocytes per 1
million cells.(>1% of all the cells in aortic arch).
The rate of leukocyte infiltration and lesion expansion
will vary with time.
Results

21. The study limitation was the possibility of immune response
interference , because highly inbred strains of rabbits are
not available, and monocyte transfusion should be done
between allogenic animals.
In another study by the same group, the above mentioned
problem was overcome by the following method.
(Arteriosclerosis Thromb Vasc. Biol. August 2000)
They transfused monocytes from a male donor into a female
recipient and then used PCR to amplify the Sry gene
(testis-determining gene) on the Y chromosome.

22. The animal model was and LDL receptor deficient mice.
Males and females from the same highly inbred strain
can be used as donors and recipients, so that no significant
immune response will occur.
Method
LDL receptor negative mice were obtained. Females who
were to be recipient , were fed an atherogenic diet, for 3-6
months, beginning at 6 to 9 months of age.
All animals showed lesions in the aortic arch covering 13%
to 49% of the total surface area.

23. Methods
Monocytes collected from male mice, injected into 3 female
mice as control and 3 female mice whom also received
cytokine injection into the peritoneum. (TNF-alpha, IL-1)
Twenty four hour later the animals were euthanized and
aorta obtained for the study.

24. Results
Figure 4. Effects of combined injection of TNF- and IL-1ß on recruitment of circulating
monocytes to aortic arch of LDL receptor–deficient mice. The experimental animals were
given 0.2 µg each of TNF- and IL-1ß in 0.5 mL of saline intraperitoneally 30 minutes after
receiving a transfusion of donor male monocytes; the control animals received only the
saline. In total, 18 animals were studied, 6 on each of 3 separate days. To take account of the
possible variability in monocyte preparations, the values for the 3 experimental animals on each
experimental day (with use of a single monocyte preparation) were expressed relative to the
mean value in the 3 control animals set equal to 100%. The mean value was 100.0±28.3% for the
9 control animals and 217.5±57.6% for the 9 experimental animals (P<0.0005) by competitive
PCR (A), and the respective values were 100.0±29.5% and 195.5±76.2% (P<0.01) by real-time
PCR (B).

25. Figure 5. Monocyte recruitment to aortic arch expressed relative to the extent of atherosclerotic
lesions. Results for control animals (•) and for cytokine-treated animals ( ) are separately plotted.
Values for cytokine-treated animals are expressed relative to the mean value in control animals set
to 100%. Note that there was almost no detectable cytokine effect in the animals with the more
extensive lesions, whereas monocyte recruitment was doubled or tripled in the animals with less
extensive lesions.
Results

26. Results
Cytokine treated mice showed 100% more recruitment
Of monocytes in the aortic wall, compared to the control
Group.
There was no cytokine effect in the animals with >40%
Of the aortic arch covered by lesions.
The data in the control animals show that monocyte
recruitment continues even when 30% to %50 of the
aortic surface is covered by lesions but that the rate of
recruitment is somewhat slower than it is in the earlier
stages of the disease.

27. SPIO is engulfed by monocyte/macrophage
System upon entry into the body.
Could it be used to detect the dynamic of
macrophage involvement in the atherosclerotic
plaque?

28. IRON H&E
Hypercholesterolemic Rabbit, Aorta, 4 days after SPIO injection

29. IRON H&E
Hypercholesterolemic Rabbit, Aorta, 4 days after SPIO injection

30. IRON H&E
Hypercholesterolemic Rabbit, Aorta, 10 days after SPIO injection

31. Hypercholesterolemic Rabbit, Aorta, 10 days after SPIO injection
H&E IRON

32. Hypercholesterolemic Rabbit, Aorta, 10 days after SPIO injection
IRON H&E

33. Hypercholesterolemic Rabbit, Aorta, 10 days after SPIO injection
IRON H&E