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Introducing a novel model

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Introducing a novel model

  1. 1. Introducing a Novel Model for StudyingIntroducing a Novel Model for Studying Macrophage Homing intoMacrophage Homing into Atherosclerotic Plaque in Apo E-Atherosclerotic Plaque in Apo E- Deficient MiceDeficient Mice Silvio Litovsky, MD, Philip Wyde, MD, WardSilvio Litovsky, MD, Philip Wyde, MD, Ward Casscells, MD, James Willerson, MDCasscells, MD, James Willerson, MD Morteza Naghavi, MDMorteza Naghavi, MD Texas Heart Institute and University ofTexas Heart Institute and University of Texas-Houston, Houston, USATexas-Houston, Houston, USA European Society of Cardiology CongressEuropean Society of Cardiology Congress Berlin, Sep 2Berlin, Sep 2ndnd , 2002, 2002
  2. 2. Why Study Monocyte Recruitment?Why Study Monocyte Recruitment?  Atherosclerosis is anAtherosclerosis is an inflammatoryinflammatory disease.disease.  Inflamed foci are associated with heavyInflamed foci are associated with heavy traffic of immune cellstraffic of immune cells mainly monocytes.mainly monocytes.  Monocytes/Macrophages are key playersMonocytes/Macrophages are key players not only in plaquenot only in plaque initiationinitiation but also inbut also in plaqueplaque progression and complicationsprogression and complications through several mechanisms.through several mechanisms.
  3. 3. MonocyteMonocyte Recruitment intoRecruitment into AtheroscleroticAtherosclerotic PlaquesPlaques Available QuantificationAvailable Quantification MethodsMethods
  4. 4. Studies done by:Studies done by:  GerrityGerrity et alet al  SteinbergSteinberg et alet al  WillersonWillerson et alet al  The potential role of SPIOThe potential role of SPIO
  5. 5. Initial EM (electron microscopy) observation by Gerrity et all (Artery 1980;8:208-14), led to the hypothesis of “macrophage assisted lipid clearance mechanism”. 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.
  6. 6. EM studies indicated bi-directional movement of foam cells through the endothelium overlying the fatty streak lesion. 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, the seems to be a traffic jam later. Fibrous cap and increased apoptosis play a role.
  7. 7. 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.
  8. 8. 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.
  9. 9. 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 into a female recipient and PCR was used to amplify the Sry gene (testis-determining gene) on the Y chromosome. They also injected cytokines to enhance plaque Inflammation.
  10. 10. 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 showed that monocyte recruitment continued even when 30% to 50% of the aortic surface was covered by lesions, but the rate of recruitment was slower than it was in the earlier stages of the disease.
  11. 11. Bottom line:Bottom line:  They are either quantitative orThey are either quantitative or qualitative.qualitative.  Although very informative, all theseAlthough very informative, all these techniques are invasive andtechniques are invasive and therefore cannot be usedtherefore cannot be used sequentially in the samesequentially in the same experimental animalexperimental animal..
  12. 12. What is SPIO?What is SPIO?  Super Paramagnetic Iron Oxide in form ofSuper Paramagnetic Iron Oxide in form of nano-particles (10-100nm).nano-particles (10-100nm).  Cored by iron oxide coated by a variety ofCored by iron oxide coated by a variety of coating materials mainly polysaccharide.coating materials mainly polysaccharide.  SPIO nano-particles are avidly taken up bySPIO nano-particles are avidly taken up by macrophages.macrophages.  Strong contrast enhancement effect inStrong contrast enhancement effect in MRI.MRI.
  13. 13. OBJECTIVEOBJECTIVE  Work from our group and other laboratories hasWork from our group and other laboratories has shown that the SPIO administered intravenouslyshown that the SPIO administered intravenously 5 to 7 days before magnetic resonance imaging5 to 7 days before magnetic resonance imaging localizes to aortic atherosclerotic plaques of apoElocalizes to aortic atherosclerotic plaques of apoE k/o mice in addition to the reticuloendothelialk/o mice in addition to the reticuloendothelial system (RES).system (RES).  In the present studyIn the present study • 1) we propose SPIO as a tool to both1) we propose SPIO as a tool to both quantitatively (iron mass spectrometry)quantitatively (iron mass spectrometry) and qualitatively (iron staining) evaluateand qualitatively (iron staining) evaluate macrophage homing into atheroscleroticmacrophage homing into atherosclerotic plaque.plaque. • 2) we study evaluate the effect of2) we study evaluate the effect of cytokine on macrophage homing intocytokine on macrophage homing into
  14. 14. FL-labeled SPIO Incubated Macrophages 24hr
  15. 15. Double DAPI Staining with Fluorescence-labeled SPIO Macrophages after 24hr Incubation
  16. 16. METHODSMETHODS  Eleven apoE k/o retired breeders, 11-Eleven apoE k/o retired breeders, 11- months old, were divided in 2 groups. Sixmonths old, were divided in 2 groups. Six received TNF-received TNF-αα 0.20.2μμg, IL-1g, IL-1ββ 0.20.2μμg andg and IFNIFNγγ 100U/g intraperitoneally, the latter100U/g intraperitoneally, the latter for 5 days; the five control received 0.5for 5 days; the five control received 0.5 mL saline containing 1% BSA. Three hoursmL saline containing 1% BSA. Three hours later, all the animals were injected withlater, 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 wereSeven days later, the recipients were euthanized, the heart and aorta perfusedeuthanized, the heart and aorta perfused under physiologic pressure.under physiologic pressure.
  17. 17. ApoE K/O mice withApoE K/O mice with SPIO but withoutSPIO but without CytokineCytokine H&E Iron Stain
  18. 18. APOE K/O Mice with SPIO andAPOE K/O Mice with SPIO and CytokinesCytokines H&E Iron Stain
  19. 19. Iron Deposition on the Edges ofIron Deposition on the Edges of Atherosclerotic PlaquesAtherosclerotic Plaques H&E Iron Stain
  20. 20. Iron Deposition in Endothelial CellsIron Deposition in Endothelial Cells
  21. 21. IMMUNOHISTOCHEMISTRYIMMUNOHISTOCHEMISTRY MAC Stain CD3 Stain
  22. 22. 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
  23. 23. Histopathologic studies of Thoracic aorta in Watanabe Hereditary Hypercholesterolemic rabbit after SPIO injection H&E staining Iron staining Mac staining
  24. 24. Histopathologic studies of Thoracic aorta in Watanabe Hereditary Hypercholesterolemic rabbit after SPIO injection H&E staining Iron staining Iron staining Iron particles
  25. 25. Quantitative AssayQuantitative Assay 270 280 290 300 310 320 330 340 350 Cytokine treated aorta Iron Content  Iron content wasIron content was higher in cytokine-higher in cytokine- treated mice thantreated mice than in sham-treatedin sham-treated apo E K/O mice:apo E K/O mice: Mass Spectrometry
  26. 26. Conclusion:Conclusion:  SPIO allows both quantitative (massSPIO allows both quantitative (mass spectrometry) and qualitative (ironspectrometry) and qualitative (iron and macrophage staining) detectionand macrophage staining) detection of infiltration of iron-ladenof infiltration of iron-laden macrophages in the aortic plaques ofmacrophages in the aortic plaques of apo E-deficient mice.apo E-deficient mice.  This effect is significantly enhancedThis effect is significantly enhanced after injection of proinflammatoryafter injection of proinflammatory cytokines.cytokines.
  27. 27. IMPLICATIONS:IMPLICATIONS:  Since SPIO is a MRI contrast agent,Since SPIO is a MRI contrast agent, SPIO-enhanced MRI may thereforeSPIO-enhanced MRI may therefore be useful for noninvasive monitoringbe useful for noninvasive monitoring of monocyte recruitment intoof monocyte recruitment into atherosclerotic plaques.atherosclerotic plaques.
  28. 28. MR Image of Abdominal Aorta After SPIO Injection in ApoE and Control Mice ApoE deficien t 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

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