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Interleukin 1, tissue necrosis factor-, and interferon- increase spio accumulation

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Interleukin 1, tissue necrosis factor-, and interferon- increase spio accumulation

  1. 1. Results • In the sham-treated apo E K/O controls, iron-positive macrophages were deposited in the subendothelial surface of the atherosclerotic plaques (Figure 1, A&B). • In the cytokine-treated mice, however, iron-positive macrophages were much more numerous and tended to localize in 2 places. In all 6 cases, the iron-laden macrophages localized in the subendothelium of plaques where they exhibited large and foamy cytoplasms. In one section of two animals, they also penetrated deeper into the plaque overlying advanced and calcified plaques (Figure 1, C&D). • In three mice, iron-laden macrophages localized in a thin layer over the internal elastic lamina, suggesting their recruitment into areas previously free of macrophage infiltration at the edges of atherosclerotic plaques (Figure 2A & B). This phenomenon was seen in sham-treated apo E K/O controls in a single case. • Although iron staining is useful, it was not uncommon to observe numerous brown golden particles in foamy macrophages on H&E staining (Figure 2A). No iron deposition was seen in the aortas of the wild-type control mice even after injection of cytokines. • In a single cytokine-treated mouse, iron-laden macrophages were also present in a single subendothelial layer in plaque-free areas (Figure 2C). • Iron-positive cellularity was greater in cytokine-treated mice than in sham-treated controls (42±11.8 vs. 11.6±5.9 iron particles/cross-section) (P< 0.0001). • Immunohistochemistry showed that most cells in atherosclerotic plaques were positive for the macrophage stain Mac-2 and a few for the T lymphocyte marker CD3 (Figure 3). • One cytokine-treated mouse exhibited a prominent mural infiltration of an intramural (muscular) coronary artery by foamy macrophages. The surrounding myocardium showed healing injury with mononuclear infiltration and early fine fibrosis (Figure 4). • Iron concentrations were higher in cytokine-treated mice than in sham-treated apo E K/O controls: 345 vs. 298 ìg/g in the aorta and 11,818 vs. 7,174 ìg/g in the liver. (Figure 5). •Electron micrograph studies have shown that iron is not distributed homogeneously in atherosclerotic plaques, but mainly subendothelially and on rare endothelial cells. (Figure 6). Interleukin-1â, Tissue Necrosis Factor-á, and Interferon-ã Increase SPIO Accumulation in Atherosclerotic Plaques of Apo E Deficient Mice Silvio Litovsky, MD, Mohammad Madjid, MD, Ward Casscells, MD, James T. Willerson, MD, Morteza Naghavi, MD Center for Vulnerable Plaque Research Texas Heart Institute & University of Texas at Houston Material and Methods •Eleven female apo E knockout (K/O) mice, each 11 months old, were divided into 2 groups. Six received tissue necrosis factor-á (0.2 µg once), interleukin-1b (0.2 µg once), and interferon-ã (100 U/g/day for 5 days) intraperitoneally; 5 received 0.5 mL saline containing 1% bovine serum albumin and served as sham-treated atherosclerotic controls. •Two wild-type C57BL/6 mice served as sham-treated nonatherosclerotic controls. •Three hours after initial cytokine or sham treatment, all mice received SPIO by intravenous injection (Feridex; Berlex Laboratories, Wayne, NJ) (1 mmol/kg iron, undiluted, injected over 3 minutes). •Six days after initial cytokine or sham treatment, recipient mice were euthanized with CO2, and their hearts and aortas were perfused under physiological pressure, and studied histologically. •In each case, the entire aorta from the sinuses of Valsalva up to the iliac bifurcation was formalin-fixed and serially sectioned transversely every 3 mm and stained with hematoxylin and eosin (H&E). • Prussian blue and MAC 2 (Accurate Chemical, Westbury, NY) stains were used for detection of iron particles and macrophages, respectively. • Total iron concentration (ìg/g of dry weight) was determined by acid digestion of plaque followed by inductively coupled plasma/mass spectrometry using a Perkin-Elmer ELAN 6100 series instrument at the Mayo Clinic Laboratories in Rochester, MN on the aortas and livers of 2 cytokine-treated and 2 sham- treated Apo E K/O. • Student’s t test was used to test for statistical significance of the difference observed in number of iron-positive cells in cytokine and sham treated animals. Introduction • SPIO (superparamagnetic iron oxide) is an MRI contrast agent in the form of nanoparticle. SPIO is avidly taken up by monocyte /macrophages in the reticuloendothelial system. •We have previously studied SPIO enhanced MR imaging of inflammation in atherosclerotic plaques of mice and rabbits. • Administration of pro-inflammatory cytokines has been shown to enhance the recruitment of monocytes into atherosclerotic lesions. • The purpose of this study was to compare the uptake of SPIO into plaques of cytokine-treated Apo E knockout (K/O) mice and age- matched, sham-treated controls in order to: 1) assess the effect of cytokines on monocyte recruitment, and 2) validate SPIO as a marker of monocyte recruitment into atherosclerotic plaques. Conclusions • Iron particles were detected subendothelially in aortic specimens from apo E K/O mice, 6 days following intravenous SPIO. This effect was much greater in mice that received cytokines intraperitoneally. • The iron was mostly present in atherosclerotic plaques, though most of the plaque area was free of iron because of the iron’s predominant superficial localization. There were, in addition, thin areas of iron-laden macrophages lying just above the internal elastic lamina, suggesting that these areas were free of monocyte infiltration before the administration of the cytokines and SPIO. Most of these zones were at the edge of atherosclerotic plaques. •No iron deposition was seen in the aortas of wild-type nonatherosclerotic mice. • Most of the iron-laden macrophages in the cytokine-treated mice were newly recruited monocytes. • SPIO also homes on new fatty streaks in the aorta of animals pretreated with cytokines. • The iron staining and mass spectrometry offer two qualitative and quantitative method for studying plaque inflammation in animal model of atherosclerosis, respectively. • Since SPIO is a contrast agent used in MRI, this may prove useful as a noninvasive method for studying monocyte recruitment into the atherosclerotic plaque in vivo. Figure 1. A. Two aortic atherosclerotic plaques in a control Apo E deficient mouse (H&E stain x40). Foamy macrophages constitute the majority of the cell population (arrows). B. Iron staining shows both plaques with faint iron particles (arrows, Prussian Blue x40). C. Advanced plaque, largely denuded of endothelium, with significant fibrosis and calcification. The intima is mainly occupied by foamy macrophages containing brown particles (arrow, H&E staining x40). D. Iron staining confirms the brown particles correspond to iron (arrow). Almost all the iron is intracellular, suggesting active uptake and not just diffusion into permeable plaque and binding to matrix. Moreover, endothelial cells contain very little stain compared to macrophages. Very little iron is present deeper in the plaque even in macrophages. (Prussian Blue x40). Figure 2. A. Thin layer of foamy cells occupies the intima on the edge of an atherosclerotic plaque (arrow, H&E x40) B. Iron staining confirms the presence of abundant iron (arrows, Prussian Blue x40). C. Area without atherosclerotic plaque shows a single layer of subendothelial iron-laden macrophages (arrow, Prussian Blue x40). Iron appears also in endothelial cells. Figure 3. A. Macrophage (MAC-2) staining confirms the phenotype of the cells in the subendothelium (arrows, MAC-2 x40). Rare cells appear to be lymphocytes (arrow head). B. Although a clear minority, some of the plaque cells were T lymphocytes as shown by the CD3 staining (arrows, CD3 x40). Figure 4. A. Intramural coronary artery with slit-like lumen and significant wall inflammation, mostly present in the media and adventitia (arrow, H&E x40). The intima appears edematous and no luminal thrombus is seen. B. The myocardium surrounding the abnormal vessel shows an organizing myocardial infarction (arrows, H&E x40). Figure 5. Comparison of iron content in aorta (A) and liver (B) (µg/g dry weight) in cytokine-treated versus sham-treated (control) mice. Figure 6. A. Electron micrograph of thoracic aorta of a WHHL rabbit injected with SPIO (1 mmol/kg iron) 5 days before sacrifice. Multiple iron particles are present in subendothelial foamy macrophages (arrows). The iron particles are about 0.5-1 micron (x4000). B. High magnification of iron particles (arrow) in macrophage lysosomes (x40,000). Texas Heart Institute® A DC C BA A CBA B

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