Introducing a Novel Model for Studying Introducing a Novel Model for Studying Macrophage Homing into Macrophage Homing into

Atherosclerotic Plaque in Apo E-Atherosclerotic Plaque in Apo E-Deficient MiceDeficient Mice

Silvio Litovsky, MD, Philip Wyde, MD, Ward Silvio Litovsky, MD, Philip Wyde, MD, Ward Casscells, MD, James Willerson, MD Casscells, MD, James Willerson, MD

Morteza Naghavi, MDMorteza Naghavi, MD

Texas Heart Institute and University of Texas Heart Institute and University of Texas-Houston, Houston, USATexas-Houston, Houston, USA

European Society of Cardiology CongressEuropean Society of Cardiology CongressBerlin, Sep 2Berlin, Sep 2ndnd, 2002, 2002

Why Study Monocyte Recruitment?Why Study Monocyte Recruitment?

Atherosclerosis is an Atherosclerosis is an inflammatoryinflammatory disease.disease.

Inflamed foci are associated with heavy Inflamed foci are associated with heavy traffic of immune cellstraffic of immune cells mainly monocytes. mainly monocytes.

Monocytes/Macrophages are key players Monocytes/Macrophages are key players not only in plaquenot only in plaque initiation initiation but also in but also in plaque plaque progression and complicationsprogression and complications through several mechanisms.through several mechanisms.

Monocyte Monocyte Recruitment into Recruitment into Atherosclerotic Atherosclerotic

PlaquesPlaquesAvailable Quantification Available Quantification

MethodsMethods

Studies done by:Studies done by: Gerrity Gerrity et alet al

Steinberg Steinberg et alet al

Willerson Willerson et alet al

The potential role of SPIOThe potential role of SPIO

Initial EM (electron microscopy) observation by Gerrity et all (Artery 1980;8:208-14), led to the hypothesis of “macrophage assisted lipid clearancemechanism”.

The swine model of hypercholesterolemia was employed. Yorkshire pigs were fed high cholesterol diets and sacrificed at the ages of 12, 15 and 30 weeks. Samples of aortic arch areas were examined by electron microscopy.

EM studies indicated bi-directional movement of foam cells through the endothelium overlying the fatty streak lesion.

The above mentioned phenomenon may explainthe stability of fatty streaks for a period of time.

It also explains that macrophages may initiallyserve as carriers of lipid material out of the plaque.

However, the seems to be a traffic jam later. Fibrous cap and increased apoptosis play a role.

In a 2nd study by Gerrity et al (Atherosclerosis 1988;71:17-25) an additional method to trace macrophages was used:

Monocytes were isolated from peripheral blood of pigs with Hypercholesterolemia and labeled with FITC (fluorocin Isothiocyanate 1-hydrochloride). Labeled monocytes were reinjected back into the animal.

1)FITC-labeled monocytes were found adherent to the thickened site of the intima but not to normal areas.

2)Labelled cells were also found within the atherosclerotic lesions.

In the study by S. Patel, James T. Willerson and Edward Yeh, (Circulation 1998;97:75-81), wild-type mice peritoneal macrophages were labeled with fluorescent latex microspheres and IV injected into ApoE deficient mice. Antibodies to ICAM-1, integrin and E-selectin were injected 6-8 hours before macrophage injection.

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.

-Antibodies against ICAM-1 and integrin significantly reduced the number of macrophage homing.

Steinberg et al (ATVB 2000;20:1976-82) reported on a new method of detecting monocytes in the plaque.

Monocytes were transfused from a male donor intoa female recipient and PCR was used to amplifythe Sry gene (testis-determining gene) on the Y chromosome.

They also injected cytokines to enhance plaque Inflammation.

Cytokine-treated mice showed 100% more recruitmentof monocytes in the aortic wall, compared to the controlgroup.

There was no cytokine effect in the animals with >40%of the aortic arch covered by lesions.

The data in the control animals showed that monocyterecruitment continued even when 30% to 50% of the aortic surface was covered by lesions, but the rate ofrecruitment was slower than it was in the earlierstages of the disease.

Bottom line:Bottom line:

They are either quantitative or They are either quantitative or qualitative.qualitative.

Although very informative, all these Although very informative, all these techniques are invasive and techniques are invasive and therefore cannot be used therefore cannot be used sequentially in the same sequentially in the same experimental animalexperimental animal..

What is SPIO?What is SPIO? Super Paramagnetic Iron Oxide in form of Super Paramagnetic Iron Oxide in form of

nano-particles (10-100nm).nano-particles (10-100nm).

Cored by iron oxide coated by a variety of Cored by iron oxide coated by a variety of coating materials mainly polysaccharide. coating materials mainly polysaccharide.

SPIO nano-particles are avidly taken up by SPIO nano-particles are avidly taken up by macrophages.macrophages.

Strong contrast enhancement effect in Strong contrast enhancement effect in MRI.MRI.

OBJECTIVEOBJECTIVE Work from our group and other laboratories has shown Work from our group and other laboratories has shown

that the SPIO administered intravenously 5 to 7 days that the SPIO administered intravenously 5 to 7 days before magnetic resonance imaging localizes to aortic before magnetic resonance imaging localizes to aortic atherosclerotic plaques of apoE k/o mice in addition to atherosclerotic plaques of apoE k/o mice in addition to the reticuloendothelial system (RES). the reticuloendothelial system (RES).

In the present study In the present study • 1) we propose SPIO as a tool to both 1) we propose SPIO as a tool to both

quantitatively (iron mass spectrometry) and quantitatively (iron mass spectrometry) and qualitatively (iron staining) evaluate qualitatively (iron staining) evaluate macrophage homing into atherosclerotic macrophage homing into atherosclerotic plaque. plaque.

• 2) we study evaluate the effect of cytokine on 2) we study evaluate the effect of cytokine on macrophage homing into plaques.macrophage homing into plaques.

FL-labeled SPIO Incubated Macrophages 24hr

Double DAPI Staining with Fluorescence-labeled SPIO Macrophages after 24hr Incubation

METHODSMETHODS Eleven apoE k/o retired breeders, 11-Eleven apoE k/o retired breeders, 11-

months old, were divided in 2 groups. Six months old, were divided in 2 groups. Six received TNF-received TNF-αα 0.2 0.2μμg, IL-1g, IL-1ββ 0.2 0.2μμg and g and IFNIFNγγ 100U/g intraperitoneally, the latter 100U/g intraperitoneally, the latter for 5 days; the five control received 0.5 mL for 5 days; the five control received 0.5 mL saline containing 1% BSA. Three hours saline containing 1% BSA. Three hours later, all the animals were injected with later, all the animals were injected with SPIO (Feridex) 1 mMol/kg of iron. SPIO (Feridex) 1 mMol/kg of iron.

Seven days later, the recipients were Seven days later, the recipients were euthanized, the heart and aorta perfused euthanized, the heart and aorta perfused under physiologic pressure.under physiologic pressure.

ApoE K/O mice with ApoE K/O mice with SPIO but without SPIO but without

CytokineCytokine

H&E Iron Stain

APOE K/O Mice with SPIO and APOE K/O Mice with SPIO and CytokinesCytokines

H&E Iron Stain

Iron Deposition on the Edges of Iron Deposition on the Edges of Atherosclerotic PlaquesAtherosclerotic Plaques

H&E Iron Stain

Iron Deposition in Endothelial CellsIron Deposition in Endothelial Cells

IMMUNOHISTOCHEMISTRYIMMUNOHISTOCHEMISTRY

MAC Stain CD3 Stain

Histopathologic study of ApoE KO Mouse injected With SPIO (Thoracic Aorta)

CD68 staining(aortic plaque)

Iron Staining (aortic plaque) Iron Staining (coronary section)

Iron particles Iron particles

Histopathologic studies of Thoracic aorta in WatanabeHereditary Hypercholesterolemic rabbit after SPIO injection

H&E staining

Iron stainingMac staining

Histopathologic studies of Thoracic aorta in Watanabe Hereditary Hypercholesterolemic rabbit after SPIO injection

H&E staining

Iron staining Iron staining

Iron particles

Quantitative AssayQuantitative Assay

270280290300310320330340350

Cytokinetreatedaorta

IronContent

Iron content was Iron content was higher in cytokine-higher in cytokine-treated mice than treated mice than in sham-treated in sham-treated apo E K/O mice: apo E K/O mice:

Mass Spectrometry

Conclusion:Conclusion: SPIO allows both quantitative (mass SPIO allows both quantitative (mass

spectrometry) and qualitative (iron and spectrometry) and qualitative (iron and macrophage staining) detection of macrophage staining) detection of infiltration of iron-laden macrophages in infiltration of iron-laden macrophages in the aortic plaques of apo E-deficient the aortic plaques of apo E-deficient mice. mice.

This effect is significantly enhanced after This effect is significantly enhanced after injection of proinflammatory cytokines. injection of proinflammatory cytokines.

IMPLICATIONS:IMPLICATIONS:

Since SPIO is a MRI contrast agent, Since SPIO is a MRI contrast agent, SPIO-enhanced MRI may therefore be SPIO-enhanced MRI may therefore be useful for noninvasive monitoring of useful for noninvasive monitoring of monocyte recruitment into monocyte recruitment into atherosclerotic plaques. atherosclerotic plaques.

MR Image of Abdominal Aorta After SPIO Injection in ApoE and Control Mice

ApoE deficient mouse

C57B1 (control) mouse

Before Injection After Injection (5 Days )

Dark (negatively enhanced) aortic wall, full of iron particles

Bright aortic lumen and wall without negative enhancement and no significant number of iron particles

in pathology