SHAPE Society

1. Influenza Infection Exerts Prominent
Inflammatory and Thrombotic Effects
on Atherosclerotic Plaques of Apo
E-Deficient Mice
Silvio Litovsky MD, Philip R. Wyde PhD, Mohammad
Madjid MD, Adeeba Akhtar MD, Sameh Naguib MD, Mir
Said Siadaty MD, Susan Sanati MD, Ward Casscells MD,
Morteza Naghavi M.D.
Center for Vulnerable Plaque Research at the University of
Texas-Houston, and Texas Heart Institute,
Influenza Research Center, Baylor College of Medicine
Houston, Texas

2. Outline:
Infection/ inflammation and atherosclerosis
Influenza and complications of atherosclerosis
Influenza on aged Apo E-deficient mice

3. Am J Epidermiol. 1998;148,10:937-948
Introduction of antibiotics and reduction in CAD (hypothetical)

4. Am J Epidermiol. 1998;148,10:937-948
Aorta of rabbits experimentally inoculated with infectious agents
Streptococcus C. pneumoniae

6. Effect of INF-γ on the development of
atherosclerosis in young Apo E-
deficient mice.

7. Outline
Influenza and complications of atherosclerosis

13. Cir
Circulation. 2000;102:3039-30045

16. Outline
Influenza on aged Apo E-deficient mice

17. Background
• Influenza infection is associated with elevated C-reactive
protein and serum amyloid A, especially in the elderly
• HDL loses its anti-inflammatory properties and LDL
becomes more susceptible to oxidation during influenza
infection*
• Mouse is a standard model for influenza and the apo E K/O
mouse is a model for atherosclerosis
• LD50 in apo E K/O mouse is comparable to LD50 of wild
type
• Maximal viral titers in the lung occur on day 4 after
inoculation; animals usually die between days 4 - 10.
(Van Lenten, Circulation 2001;103:2283-8)

18. Methods
• 33 apo E K/O mice of either sex 2-2.5 years old and 10 age-
matched C57 BL were intranasally injected with 1LD50 of
influenza A virus
• Body weight, heart rate and O2 saturation determined at
inoculation, day 3, day 5 and at time of sacrifice
• Sacrificed at days 3, 5, 10 and 15. In case of spontaneous
death, autopsy was performed within 12 hours
• Virus quantification on homogenized lungs determined on
day 4 by hemagglutination
• RT-PCR for the presence of influenza mRNA in the aorta of
2 animals
• Aorta up to the level of the renal arteries were excised, fixed
in formalin and processed

19. Results
• Hemagglutinating virus isolated from every virus-
inoculated mouse.
• No clear evidence of influenza RNA on aortic samples
• 13 animals died between days 4 and 10. All inoculated
animals lost weight. Eleven infected
(7 sacrificed and 4 fatalities) showed striking intimal
infiltrates. Nine out of the 11 with intimal infiltrates
died or were sacrificed on day 10 +/- 1 day.

23. Smooth muscle actin
Mac-1
CD4

25. Cytokine-SPIO Protocol
•Purpose: To enhance macrophage homing to plaque and monitor
it by SPIO. Compare the iron particles present in macrophages of apo E-
deficient atherosclerotic plaques under baseline conditions (control group)
and after the administration of TNF-α, IL-1β and IFN-γ (influenza
simulated group).

26. Cytokine-SPIO Protocol
•Protocol: Eight retired female breeders, approximately 12-month old
were divided in 2 groups. Five received I.P. 0.2 µg each of mouse
recombinant TNF- and IL-1ß; and 100 units/g INF-γ for six days; the 3
control received 0.5 mL saline containing 1% BSA. Three hours later, all
the animals were I.V. injected with Feridex 1 mMol/kg of iron. Seven days
later, the recipients were euthanized with CO2 and perfused under
physiological pressure.
The entire aorta up to the iliac bifurcation was formalin-fixed, serially
sectioned transversally and stained for H&E and iron.

27. Iron Staining H&E Staining
Apo E-deficient mouse injected with SPIO
No cytokines

28. Iron Staining H&E Staining
Apo E-deficient mouse injected with SPIO
No cytokines

29. Iron Staining H&E Staining
Apo E-deficient mouse injected with SPIO
Cytokines added

30. Iron Staining H&E Staining
Apo E-deficient mouse injected with SPIO
Cytokines added

31. Apo E-deficient mouse injected with SPIO
Cytokines added
H&E Mac3 CD3

32. Intramural coronary
artery involvement
Myocardial injury
Apo E-deficient mouse injected with SPIO
Cytokines added

33. Conclusion
• Influenza A virus exerts prominent pro-inflammatory and
pro-thrombotic effects in about one third of aged apo E
K/O mice
• Studies with longer follow-up periods are necessary to
determine whether increased plaque burden and/or
aneurysm formation occur
• Significance of LD 50 dose is unclear for atherosclerosis.
Other doses are being planned
• Plaque inflammation as seen in this model has not been,
to our knowledge, previously reported in infectious
experimental models of atherosclerosis

34. Conclusion
Cytokine effect likely accounts, at least partially,
for the effects of influenza infection on the
atherosclerotic plaques

35. Acknowledgement
James T. Willerson, MD

36. Center for Vulnerable Plaque Research
Houston, Texas