BRIEF REPORT |
Intracellular Localization of Oxidized Low-density Lipoproteins in Atherosclerotic Plaque Cells Revealed by Electron Microscopy Combined with Laser Capture Microdissection
Surgical Professorial Unit, University of New South Wales, St. Vincent's Hospital, Sydney, Australia
Correspondence to: Dr. Yuri V. Bobryshev, Surgical Professorial Unit, Level 5, DeLacy Building, St. Vincent's Hospital, Sydney, Darlinghurst, NSW 2010, Australia. E-mail: y.bobryshev{at}unsw.edu.au
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Summary |
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(J Histochem Cytochem 53:793797, 2005)
Key Words: laser capture microdissection immunohistochemistry transmission electron microscopy atherosclerosis carotid arteries foam cells oxidized low-density lipoproteins
NORMAL AND PATHOLOGICALLY altered tissues consist of complex compositions of cellular populations and subpopulations. Histologic and cytologic changes are central to the diagnosis and classification of many disease processes. Recently, laser capture microdissection (LCM) of tissue sections and cytological preparations has become increasingly used for the isolation of homogeneous cell populations from tissues, thus overcoming the obstacle of tissue complexity (Fend and Raffeld 2000; Hunt and Finkelstein 2004
). In conjunction with sensitive analytical techniques, LCM allows the precise examination of rare cell populations that are otherwise inaccessible for conventional molecular studies (Fend and Raffeld 2000
; Hunt and Finkelstein 2004
). In the LCM method, a transparent film, which allows the visualization of cells microscopically, is placed over a tissue section or a cytological sample, and the film is then selectively adhered to cells of interest by a focused pulse of an infrared laser (Fend and Raffeld 2000
; Hunt and Finkelstein 2004
). The film with the adhered cells is removed from the original sample and is placed directly into DNA, RNA, or protein-extraction buffer for further processing. LCM has revolutionized molecular analysis of complex tissues by combining the power of molecular genetics, genomics, and proteomics with the topographic precision of microscopy (Fend and Raffeld 2000
; Hunt and Finkelstein 2004
).
LCM was originally designed to capture cells for genetic analysis, but there are instances where the identification of structural peculiarities of a particular cell population is required. Recently, Grant and Jerome (2002) reported a method for preparing microdissected, cultivated cells for further electron microscopic analysis. Although the method described by Grant and Jerome (2002)
provides fine structure presentation, it is limited to investigating cultivated cells. Techniques that would utilize LCM for electron microscopic investigation of tissue specimens or techniques that would enable the identification of the location of an antigen of interest within cells are not currently available.
The present work was undertaken to develop a technique suitable for the ultrastructural investigation of cells microdissected from tissue sections. In the present work, human atherosclerotic arterial tissue was used and the intracellular localization of oxidized low-density lipoproteins (ox-LDL) in plaque cells was investigated. The accumulation of ox-LDL in the arterial intima is a key event in atherosclerotic lesion development (Ross 1999; Stocker and Keaney 2004
); however, the structural mechanisms of ox-LDL accumulation in human atherosclerosis in situ have had little study and are poorly understood (Kaesberg et al. 1993
; Ross 1999
). In a previous work by Kaesberg et al. (1993)
, immunogold-labeled ox-LDL were used to show the presence of the signal within the macrophage-origin cells in human atherosclerotic plaques, but the characteristics of intracellular distribution of ox-LDL are yet to be revealed.
For the present work, atherosclerotic plaques were cut from seven carotid artery specimens obtained at endarterectomy at St. Vincent's Hospital, Sydney, from patients whose ages ranged from 52 to 71 years. The material was collected in accordance with the principles outlined in the Declaration of Helsinki of 1975. The study was approved by the institutional review board of St. Vincent's Hospital, Sydney. Plaques were cut into thin tissue slices (2-mm thick) and were fixed for 2 hr at 4C in a solution containing 4% paraformaldehyde and 0.1% glutaraldehyde in phosphate-buffered saline (PBS), pH 7.4.
After washing the tissue slices in PBS (three times for 30 min each) and pretreating them with 0.3% H2O2 and 0.5 µM butylated hydroxytoluene (Sigma; St Louis, MO), immunohistochemical reaction with ox-LDL antibody (2 µg/ml) was carried out on the "floating" tissue slices. The present work utilized ox-LDL antibody (Winyard et al. 1993), a kind gift from Dr. Franz Tatzber. The standard avidinbiotin complex technique (Hsu et al. 1981
) was used, as described previously (Bobryshev and Lord 1998
), but the periods of the incubations with both the primary and secondary antibodies were increased (12 hr each at 4C). The product reaction was visualized using 3,3' diaminobenzidine (DAB) (DAKO; Glostrup, Denmark) and, in some cases, with 3-amino 9-ethylcarbazole (AEC) substrate (DAKO). In some cases, for the enhancement of electron density of immunohistochemical reaction product, 6 mg/ml ammoniumnickel sulfate was added to DABH2O2 solution according to a method of Tago et al. (1986)
, modified by Punnonen et al. (1999)
. Negative controls were carried out as described previously (Bobryshev and Lord 1998
).
The tissue slices were then washed in PBS, refixed in 1% glutaraldehyde in PBS, orientated and embedded in OCT compound in liquid nitrogen. After cutting in a cryostat, frozen sections were placed onto object slides covered by polyethylenenaphthalate (PEN) membranes (1440-1000; P.A.L.M. Microlaser Technologies, Bernried, Germany). For microdissection, a PALM Laser-MicroBeam System (P.A.L.M. Microlaser Technologies) that enables the contact-free isolation of single cells was used. Cells containing ox-LDL as well as focal extracellular accumulations of ox-LDL were observed in all plaques studied. Cells containing ox-LDL were most frequent in areas surrounding the necrotic cores (Figure 1). Consecutive stages of the selection and capture of cells containing ox-LDL are shown in Figure 1. From several sections obtained from each plaque, up to 40 ox-LDL immunopositive cells were microdissected and catapulted into the lid of a 0.5-ml reaction tube using the laser pressure catapulting technique of the instrument. The reaction tube lid containing microdissected cells was removed from the LCM instrument, fixed in 1% glutaraldehyde in PBS (pH 7.4) for 1 hr, and postfixed in 1% osmium tetroxide. The reaction tube leads with microdissected cells obtained from different plaque specimens were embedded in Araldite resin using a routine technique (Bobryshev and Lord 1996) but with the omission of acetone (or propyleneoxide) to avoid dissolving the reaction tube lids. Consecutive steps in the collection and embedding of ox-LDL immunopositive cells are shown in Figure 2.
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Electron microscopic analysis demonstrated that the cytoplasm of microdissected cells contained membrane-bound vacuoles and membrane-free lipid vacuoles ("lipid droplets"), enabling the identification of these cells as foam cells (Figure 3A). The cytoplasm of microdissected cells was not only highly vacuolized but was also free of myofilaments, suggesting the macrophage origin of microdissected cells. ox-LDL were observed within vacuoles with the diameters ranging from 200 to 500 nm (Figures 3B and 3C). The association of ox-LDL with microvesicles of 40 to 60 nm in diameter was also observed (Figures 4A and 4B). Some microvesicles containing ox-LDL were located along the border of "lipid droplets" as well as within the peripheral portion of "lipid droplets" (Figure 4B). The study was extended by means of a double immunohistochemistry utilizing ox-LDL antibody and anti-CD68. Double immunostaining included a combination of peroxidaseanti-peroxidase and alkaline phosphataseanti-alkaline phosphatase techniques, which were carried out as described previously (Bobryshev and Lord 1998). This staining confirmed cellular colocalization of ox-LDL with CD68 antigen (Figure 4C).
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In addition to the procedures described above for the preparation of tissue specimens for further LCM, in some experiments of the present study, arterial tissue slices were immunostained using the labeled streptavidinbiotin complex technique (LSAB; DAKO) according to the manufacturer's instructions, followed by the visualization of the antigen with Fast Red substrate (DAKO). Similarly, as in the experiments utilizing AEC, the visualization of reaction product with Fast Red substrate did not lead to the formation of electron-dense reaction products. If the goal of a study requires the investigation of ultrastructural characteristics of LC-microdissected cells only, the use of Fast Red or AEC substrate may be preferable to the use of DAB. If an antigen of interest is abundant within cells, the use of AEC or Fast Red substrate would prevent an intensive masking of the ultrastructure, which might occur during the visualization of a reaction using DAB.
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Acknowledgments |
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I would like to thank Dr. Franz Tatzber, Biomedica, Vienna, Austria, for the kind gift of ox-LDL antibody.
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Footnotes |
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Literature Cited |
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