Periadventitial Fat May Play an Important Role in Plaque

Inflammation; Introducing Macrophage-Like Activity of

Adipocytes in Periadventitial FatMouse – Rabbit - Human

Silvio Litovsky, MD, Mohammad Madjid, MD, Alireza Zarrabi, MD, Ward Casscells, MD, James

T. Willerson, MD, Morteza Naghavi, MD

Center for Vulnerable Plaque Research, Texas Heart Institute, and University of Texas-

HoustonHouston, TX

Introduction• Our group has long been interested in the use of

SPIO (superparamagnetic iron oxide) nanoparticles as a contrast agent for magnetic resonance imaging of atherosclerotic plaques.

• SPIO is taken up by fixed macrophages of the reticuloendothelial system (RES) and by plaque macrophages, mainly subendothelial.

• In our MRI-histopathology correlation studies, we noted that iron was present not only inside plaque macrophages, but also in the periadventitial fat.

Method:• Twenty two female apoE K/O mice, 12 months old, and eleven

C57BL/6 female mice, 6 months old, were injected intravenously with SPIO (1 mmol/kg iron).

• Six days later, mice were euthanized, and their aortas perfusion-fixed. The entire aorta was formalin or Bouin’s-fixed and serially sectioned. Subcutaneous abdominal fat was also obtained from every animal.

• Prussian blue and MAC-2 stains were used for detection of iron particles and macrophages, respectively. The entire available periadventitial fat was analyzed from each section.

• The 6 day time point was chosen because work from our laboratory has shown that the highest MRI resolution is achieved 5-7 days after injection; corresponding histology also showed highest iron uptake around this time.

Methods

Day 0 Day 6

SPIO Injection Sacrifice and Pathology

SPIO Uptake in Periadventitial Fat

A B

A – Wild type mouse B – ApoE K/O mouse

A

MAC-2 Positive Cells in Periadventitial Fat

BB

A – Wild type mouse B – ApoE K/O mouse

F4/80 Positive Cells In Periadventitial Fat of ApoE Deficient MiceApoE K/O Mouse

Iron Stained Area in the Periaortic Fat of C57BL/6 and ApoE K/O Mice.

0200400600800

100012001400160018002000

C57BL Apo E K/O

C57BL/6 ApoE K/O p valueTotal Iron Area (µm2) / Total Fat Area (mm2) 382 ± 291 1896 ± 3847 0.032

Iron area in the subcutaneous abdominal fat of C57BL/6 and ApoE K/O mice.

050

100150200250300350400450

C57BL Apo E K/O

C57BL/6 ApoE K/O p value

Total Iron Area (µm2) / Total Fat Area (mm2) 293 ± 265 427 ± 366 NS*

Rabbit Studies

Iron Uptake In WHHL Rabbit 7 Days After Administration of SPIO

plaquemedia

adventitiaPeriadventitial fat

H&EIronStain

Iron Co-localizes With Subendothelial & Periadventitial Fat RAM11 Positive Cells

A

B

Iron Uptake In NZW Rabbit 7 Days After Administration of SPIO

H&E Iron Stain

Human Studies

CD68 in Periadventitial Fat of a Coronary Artery With Intimal Thickening

H&E

CD68

CD68 Positive Cells Are Numerous in the Plaque and Periadventitial Fat

A B

H&E CD 68

The Overwhelming Majority of CD68 Positive Cells Are Not S100 Positive

S100CD68

Toluidine blueCD68

The Overwhelming Majority of CD68 Positive Cells in the Periadventitial Fat Are Not Toluidine Blue Positive

Co-localization of CD68 and CD34 Positive Cells

CD34CD68

There is moderate co-localization of CD68 & CD34 positive cells

CD68 CD34

Conclusions

• Periaortic fat tissue in apoE K/O mice takes up SPIO nanoparticles (a MRI contrast agent).

• The magnitude of the uptake in apoE K/O mice is much greater than in C57BL/6 mice. Similarly, the number of macrophage/macrophage-like cells in the fat of apoE K/O mice is greater than in C57BL/6 mice.

• Preliminary data on WHHL and New Zealand White rabbits indicate a larger uptake of iron following SPIO in the periaortic fat of the hypercholesterolemic atherosclerotic rabbit.

Conclusions (…continued)

• Preliminary studies on human coronaries show a much greater density of CD68 positive cells in the periarterial fat of atherosclerotic vessels compared to normal.

• Immunohistochemistry indicates that these cells are not dendritic cells or mast cells. Moderate co-localization with endothelial cells was seen.

• In many cases, the border between adventitia and

fat is not well defined, especially in humans. The cellular infiltrate in the periadventitial fat was similar to the infiltrate in the adventitia in all cases, suggesting they behave as a single physiologic unit.

Conclusions (…continued)

• The exact lineage of these cells remains to be further elucidated. Fat tissue is known to contain a stroma-vascular fraction (SVF) and the cells described here in the periadventitial fat of mouse and rabbit aorta and human coronaries, show similarities with this cell type.

• The possible significance of these cells with macrophagic properties in the progression of atherosclerosis, its complications and restenosis remains to be investigated.

Conclusions (…continued)

• Upon further confirmation, these findings may introduce a new marker of plaque vulnerability based on inflammation in periadventitial fat.

• From the imaging standpoint, the large periadventitial area may provide a new opportunity in imaging vulnerable plaque where the spatial resolution for imaging fibrous cap remains a challenge.

                                                                                                                                                                      

                                    

Texas Heart Institute University of Texas-Houston

Center for Vulnerable Plaque Research