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Monocyte recruitment into atherosclerotic plaques


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Monocyte recruitment into atherosclerotic plaques

  1. 1. Monocyte Recruitment into Atherosclerotic plaques Quantification Methods
  2. 2. Review of the studies done by: • Gerrity et al • Steinberg et al • Willerson et al • The potential role of SPIO
  3. 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. 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. 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. 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. 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. 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. 9. Figure 2. Schematic diagram of the study area depicting a labeled macrophage adhering to the plaque.
  10. 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. 11. Figure: Macrophages labeled with fluorescent microspheres adhere to atherosclerotic plaques.
  12. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 28. IRON H&E Hypercholesterolemic Rabbit, Aorta, 4 days after SPIO injection
  29. 29. IRON H&E Hypercholesterolemic Rabbit, Aorta, 4 days after SPIO injection
  30. 30. IRON H&E Hypercholesterolemic Rabbit, Aorta, 10 days after SPIO injection
  31. 31. Hypercholesterolemic Rabbit, Aorta, 10 days after SPIO injection H&E IRON
  32. 32. Hypercholesterolemic Rabbit, Aorta, 10 days after SPIO injection IRON H&E
  33. 33. Hypercholesterolemic Rabbit, Aorta, 10 days after SPIO injection IRON H&E