TECHNICAL NOTE

A Novel Technique For Mapping the Lipid Composition of Atherosclerotic Fatty Streaks by En Face Fluorescence Microscopy

Correspondence to: Anne M. Klinkner, Cellular Pathology UE0462, SmithKline Beecham Pharmaceuticals, PO Box 1539, King of Prussia, PA 19406.

	Summary

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We introduce here a new fluorescence microscopy technique for en face analysis of the atherosclerotic fatty streaks (FS). This technique is semiquantitative and has the sensitivity and resolution to map lipids to individual cells in FS less than 100 µm in diameter. New Zealand White rabbits were fed an atherogenic diet for up to 26 weeks. Aortas were fixed in formalin and stained en bloc with the fluorescent dyes Nile red and filipin. Fluorescent staining was validated by correlating microfluorimetric and biochemical measurements of the lipid content in FS. To determine the cell types associated with the different staining patterns, FS were also evaluated by transmission electron microscopy (TEM) and immunohistochemistry (IH). Correlation of microfluorimetry, TEM, IH, and biochemical data indicated that regions rich in non-esterified cholesterol stained with filipin and fluoresced blue owing to accumulations of lipid vessicles and/or cholesterol crystals. Regions rich in neutral and polar lipids stained with Nile red and fluoresced yellow or orange, respectively, owing to accumulations of lipids in both macrophages and smooth muscle cells (SMC). Digital overlays of the filipin and Nile red images revealed that larger lesions (>0.5 mm diameter) had a "nested" distribution of lipids, with a blue (filipin) fringe surrounding an orange (Nile red) fringe surrounding a yellow (Nile red) center. (J Histochem Cytochem 45:743-753, 1997)

Key Words: filipin, Nile red, computer-assisted image, processing

	Introduction

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A PRIMARY effort in the understanding of atherosclerotic disease progression has been the development of methods for visualization of atherosclerotic plaques in the earliest stages of formation of the fatty streak (FS). Although our understanding of the pathogenesis of atherosclerosis has advanced dramatically over the past decade (Williams and Tabas 1995 ; Wissler 1994 ; Ross 1993 ), biochemical analysis of the earliest lesions, i.e., those less than a few millimeters in diameter, remains problematic.

Fluorescence and light microscopy have been instrumental in locating and identifying lipid components of atherosclerotic lesions in animal models and human biopsy tissues (Brown et al. 1992 ; Siefert 1989; Lupu et al. 1987 ; Fowler and Greenspan 1985 ; Kruth 1984a , Kruth 1984b , Kruth 1985 ; Kruth and Fry 1984 ). Immunohistochemical (IH) methods have been used to distinguish populations of smooth muscle cells and macrophages in atherosclerotic lesions (Masuda and Ross 1990 ; Rosenfeld et al. 1987 ; Tsukada et al. 1986 ), and many authors have used electron microscopy to characterize cell types and investigate intracellular aspects of atherosclerosis (Masuda and Ross 1990 ; Lupu et al. 1987 ; Rosenfeld et al. 1987 ). However, techniques such as these, which rely on the evaluation of tissue cross-sections, make the study of entire FS difficult. Moreover, biochemical techniques utilizing extraction procedures do not provide information about the spatial relationships of the lipids and cells composing the FS and lack the sensitivity to study the smallest FS.

We describe here a novel en face fluorescence technique that allows high-resolution mapping of the lipid composition of entire FS. Aortas were collected from New Zealand White rabbits fed an atherogenic diet. After fixation and dual fluorescent staining with filipin and Nile red, digital fluorescence microscopy was used to map areas of lipid accumulation in the aorta. Electron microscopy, immunohistochemistry, and biochemical methods were utilized to validate the staining technique and to identify the cell types and lipid deposits associated with the fluorescently stained regions.

	Materials and Methods

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Generation of Atherosclerotic Tissues
To generate the widest size range of atherosclerotic lesions, New Zealand White rabbits received an atherogenic diet (Purina Mills; St Louis, MO) of 0.05%, 0.1%, 0.2%, or 0.3% w/w for the first 6 weeks. They were then maintained on 0.15% cholesterol for 14 or 26 weeks. Plasma cholesterol was monitored weekly and averaged over time. The mean (± SD) plasma cholesterol was 596 ± 153 mg/dl for the rabbits sacrificed at 14 weeks and 431 ± 271 mg/dl for those sacrificed at 26 weeks. The rabbits were sacrificed by sodium pentobarbital injection and exsanguination. The aorta was removed, opened lengthwise, pinned out on cork boards, and fixed with phosphate-buffered formalin.

Nile Red/Filipin Fluorescent Staining
Stock solutions of Nile red (Sigma Chemical; St Louis, MO) (0.5 mg/ml in acetone) and filipin (Sigma) (2.5 mg/ml in dimethyl formamide) were prepared and stored at -20C. Pieces of aorta (0.5 x 1.0 cm) containing FS ranging from barely visible to >50 mm², to those that occupied the entire circumference of the aorta, were trimmed and the adventitia removed by sharp dissection. The pieces were then washed three times for 15 min in PBS w/o Ca⁺² or Mg⁺², incubated in Nile red/filipin (100 µl Nile red stock + 200 µl filipin stock in 10 ml PBS) for 30 min, washed in PBS three times for 30 min, rinsed with distilled water, laid flat on glass slides, and coverslipped with Crystal Mount (Biomedia; Foster City, CA).

Fluorescence Microscopy
Stained aortas were examined with a Leitz Orthoplan light microscope equipped for epifluorescent illumination. The following filters were used to view stained aortas: 390-490-nm bandpass excitation and 530-550-nm cutoff barrier for Nile red and 340-380-nm bandpass excitation and 430-500-nm cutoff barrier for filipin. Images were recorded digitally from red-green-blue (RGB) video signals generated with a TEC-470 CCD video camera (Optronics Engineering; Golata, CA). RGB images were converted to hue-saturation-intensity (HSI) signals and the hue spectra for each lesion were plotted with ImagePro Plus software (Media Cybernetics; Silver Spring, MD). Finally, hue values were converted to wavelengths (nm) with a standard curve generated with gelatin Wrattan filters (Eastman Kodak; Rochester, NY). Nile red stained neutral lipids yellow (580-596 nm, 590-nm peak fluorescence) and polar lipids orange (597-620 nm, 600-nm peak fluorescence). Filipin-stained non-esterified cholesterol fluoresces blue (460-480 nm, 470-nm peak fluorescence). The Nile red and filipin images were overlaid digitally using OPTIMAS software (OPTIMAS; Edmonds, WA) to create composite images.

Microfluorimetry and Lipid Analysis by Thin-layer Chromatography
To correlate fluorescence intensity with biochemical measurements of lipid content of the lesioned aorta, 4-mm-diameter punch biopsies were taken from the aortas of rabbits after 14 weeks on the atherogenic diet and were imaged for Nile red/filipin staining (x 2 objective) as described above and subsequently processed for lipid analysis. The RGB signals were converted to HSI signals using Image Pro Plus software, the images thresholded for specific emission wavelengths and the fluorescence intensity for putative neutral lipids (Nile red emission 570-590 nm), polar lipids (Nile red emission 590-620 nm), and non-esterified cholesterol (filipin emission 460-500 nm) was measured in arbitrary units. After imaging, the punch biopsies were washed in distilled water, blotted, and subjected to Folch extraction [2:1 choloroform:methanol (v/v)] (Folch et al. 1957 ). The extracts were dried under nitrogen, brought up in a known volume, and spotted onto sintered silica rods (Chromorods, Type SIII ) for thin-layer chromatography/flame ionization detection (TLC/FID). TLC were developed with chloroform:methanol:water (45:20:2) twice for 3 min, followed by hexane:diethyl ether:formic acid (60:5:0.5) for 36 min. Lipids were measured using an Iatroscan MK-5 TLC/FID Analyzer (RSS; Costa Mesa, CA) and were quantitated against standard curves generated with known lipid standards (Sigma). Fluorimetry and biochemical data were plotted as scatter diagrams and linear regression analysis was performed using Table Curve software (Jandel Scientific; Corte Madera, CA). The statistical significance of the correlations was tested by student's t-test; p<0.05 was accepted as significant.

Immunohistochemistry
Paraffin sections of aorta containing lesions similar in size and location to those stained fluorescently were stained with either anti-smooth muscle -actin monoclonal antibody ASM-1 (Boehringer Mannheim Biochemicals; Indianapolis, IN) or anti-rabbit alveolar macrophage monoclonal antibody RAM11 (Dako; Carpinteria, CA) for detection of smooth muscle cells and macrophages. The other reagents were purchased as a kit and staining was performed on a TechMate Automated Staining System (BioTek Solutions; Santa Barbara, CA). Serial 5-µm sections were cut from paraffin-embedded segments of aorta, mounted on Capillary Gap Plus slides (BioTek Solutions), and air-dried. Slides were deparaffinized, rehydrated, and steamed for 20 min in heat-induced epitope retrieval buffer. Endogenous peroxidase was blocked with 3% H₂0₂ in buffer for 15 min. Nonspecific binding was blocked with normal goat serum in PBS buffer for 15 min. The tissue sections were incubated with specific antibodies for 4 hr at room temperature and developed with biotinylated secondary antibodies (ABC) and peroxidase-conjugated streptavidin, using diaminobenzidine as the chromogen. Slides were then counterstained with hematoxylin, dehydrated, cleared, and coverslipped using a xylene-based mounting medium.

Transmission Electron Microscopy
Lesions similar in size and location to those stained fluorescently were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer, rinsed in buffer, postfixed in 2% buffered osmium tetroxide for 4 hr, dehydrated in a graded series of ethanols, and embedded in Embed 812 (Electron Microscopy Sciences; Ft Washington, PA). Semithin sections were cut on a Rei-chert Ultracut E, stained with toluidine blue, and lesions selected for thin sectioning. Thin sections were stained with uranyl acetate and lead citrate and examined in a JEOL 1200EX transmission electron microscope.

	Results

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Punch Biopsies
To ensure that the fluorescent staining accurately reflected the lipid composition of the lesions, punch biopsies (4-mm diameter) from aortas from rabbits collected after 14 weeks on the atherogenic diet were stained with Nile red and filipin, and fluorescent images were stored digitally. Examples of the images for a biopsy are shown in Figure 1A and Figure 1B. Histograms of the fluorescence intensity of Nile red and filipin emission vs wavelength (Figure 2A and Figure 2B) were generated. In the Nile red histogram, identifiable peaks at 600 nm (orange) and 593 nm (yellow) divided the histogram into two regions. The histogram of the filipin emission was monomodal, with a peak at 468 nm (blue).

View larger version (114K): [in this window] [in a new window]   Figure 1. Fluorescence microscopy of fatty streaks after staining with Nile red and filipin. (A) Filipin fluorescence image of a punch biopsy from an aorta collected after 14 weeks on an atherogenic diet. (B) Nile red fluorescence image of the same punch biopsy shown in A. Punch biopsy is 4 mm diameter, x2 original magnification. (C) Filipin fluorescence image of an area of aorta collected after 26 weeks on an atherogenic diet, x2 original magnification. The smallest discernible lesions are 0.01-0.2 mm2 in size and stain solely with filipin. (D) Nile red fluorescence of the same area shown in C. Areas of orange or yellow fluorescence reflect the lipid compositions of those regions (yellow, non-polar; orange, polar). (E) Digitally overlaid Nile red and filipin images. Larger lesions (0.2-1.0 mm2) have blue (filipin) fringes with a central region stained orange with Nile red. The largest lesions studied (>2 mm2) had concentric blue and orange fringes surrounding a central region that stained yellow with Nile red. (F) Digitally overlaid Nile red and filipin images at higher magnification. Individual cell profiles (*) can be discriminated at the edge of the lesion. x10 original magnification. Bars: A-E = 500 µm; F = 100 µm.

View larger version (34K): [in this window] [in a new window]   Figure 2. Histograms showing fluorescence vs emission wavelength of filipin (excitation wavelength 340-380 nm) (A) and Nile red (excitation wavelength 450-590 nm) (B) of the punch biopsies shown in Figure 1A and Figure 1B, respectively. (C) TLC/FID chromatogram from the biopsy in Figure 1A and Figure 1B. CE, cholesteryl ester; C, cholesterol phospholipids; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PC, phosphatidylcholine; SM, sphingomyelin; LPC, lysophosphatidylcholine.

Lipids were extracted from 11 punch biopsies after fluorescence spectral analysis and the lipid composition was analyzed by TLC/FID. Figure 2C is the chromatogram from the biopsy illustrated in Figure 1A and Figure 1B and quantitated by spectral analysis in Figure 2A and Figure 2B. Major peaks from cholesteryl-esters (CE), non-esterified cholesterol (C), and phospholipids (PL) were quantitated. Neither Nile red nor filipin was found to interfere with TLC/FID. Correlation analysis of fluorescence intensity and lipid composition is shown in Figure 3. In general, larger lesions had higher levels of fluorescence at all three wavelength ranges evaluated. As expected, larger lesions also tended to have a higher content of CE, C, and PL. In several smaller FS, Nile red and filipin fluorescence were measurable when the lipid content was below the limit of detection of TLC-FID (range 0.05-0.5 µg/biopsy, depending on lipid class), as evidenced by points plotted on the y-axis in Figure 3. Statistically significant correlations were found between filipin fluorescence and C content and between Nile red yellow fluorescence and CE content. Filipin therefore detects regions rich in non-esterified cholesterol and Nile red yellow detects regions rich in esterified cholesterol. Despite the lack of correlation with Nile red orange fluorescence (580-600 nm), a significant correlation was found between total Nile red fluorescence (580-620 nm) and total non-cholesterol lipid (CE+PL) content. This suggests that Nile red orange fluorescence detects areas of mixed lipid composition that are relatively polar in nature and therefore are probably enriched in phospholipids.

View larger version (16K): [in this window] [in a new window]   Figure 3. Correlation analysis of fluorescence intensity and lipid composition. (A) Non-esterified (free) cholesterol vs filipin fluorescence (r2=0.76; p<0.01). (B) Cholesteryl esters vs Nile red yellow fluorescence (r2=0.90; p<0.001). (C) Total non-cholesterol lipids (CE+PL) vs total Nile red fluorescence (yellow + orange) (r2=0.76; p<0.01).

Early Stages of FS Formation
For detailed study of the lipid and cellular composition of developing FS, aortas were collected from five rabbits after 26 weeks on an atherogenic diet and were stained with Nile red and filipin. Adjacent areas were processed for immunohistochemistry and transmission electron microscopy to study cell composition or were observed by fluorescence microscopy before staining. This tissue provided areas of normal aorta, very small FS, and well-developed FS, which occupied the entire circumference of the aorta.

The smallest lesions observed were isolated areas (0.01-0.2 mm²) that stained solely with filipin. There was little or no autofluorescence in the emission range of filipin. Filipin staining at the edges of these lesions was ragged and feathery (Figure 1C). TEM of these regions revealed phospholipid vesicles (Figure 4A) in intimal spaces beneath an intact endothelium. In larger lesions, filipin staining was globular and brighter towards the center of the lesion (Figure 1C). TEM revealed cholesterol crystal clefts among densely packed aggregates of lipid vesicles at the bases of larger (>1-mm²) lesions (Figure 4B).

View larger version (189K): [in this window] [in a new window]   Figure 4. Transmission electron microscopy (TEM) of a lesion collected after 26 weeks on an atherogenic diet. (A) Intercellular space filled with phospholipid liposomes (pl) in intimal spaces at a lesion edge. Similar deposits were observed in the smallest (0.01 to 0.2 mm2), pre- lesional regions that stained with filipin alone. Bar = 1 µm. (B) Intercellular space containing cholesterol crystal clefts (cc) and densely packed lipid liposomes (l) from the base of a larger (>1-2 mm) lesion. c, dollagen fibrils; e, elastin. Bar = 1 µm.

The smallest FS that stained with Nile red was as minimal as a few cells in diameter (0.2-1 mm²) and fluoresced orange (Figure 1D). There was little or no autofluorescence in the emission range of Nile red. Staining at the perimeter of larger lesions was also orange, and occasionally corresponded with individual cell profiles (Figure 1F, asterisk). Immunohistochemical studies of small lesions showed positive staining for both macrophages and smooth muscle cells (Figure 5C and Figure 5D). TEM revealed macrophages (Figure 5A) and smooth muscle cells (Figure 5B), with many secondary lysosomes containing phospholipid-rich inclusions. Smooth muscle cells were distinguished from macrophages by nuclear chromatin pattern, cytoplasmic fibers, myofilaments, and the presence of characteristically larger lipid inclusions. Cholesterol crystal clefts were seen occasionally in lysosomal bodies of foam cells (Figure 5A).

View larger version (133K): [in this window] [in a new window]   Figure 5. Transmission electron microscopy (TEM) of a lesion collected after 26 weeks on an atherogenic diet, showing cells from a region of orange fluorescence by Nile red. (A) Macrophage with non-membrane-bound neutral lipid droplets (l), phospholipid inclusions in secondary lysosomes (pl), and a cholesterol crystal cleft (cc). Bar = 100 µm. (B) Smooth muscle cell containing many phospholipid rich inclusions (pl) and neutral lipid droplets (l). Bar = 1 µm. Immunohistochemical staining of Nile red orange areas for (C) macrophages and (D) smooth muscle cells. Bars = 100 µm.

After Nile red staining, in most lesions larger than 1 mm² the center fluoresced yellow, surrounded by an orange fringe (Figure 1D). Yellow staining tended to be globular and occasionally reflective of individual cell profiles. IH indicated that larger lesions were composed primarily of macrophages and contained few smooth muscle cells (Figure 6C and Figure 6D). Macrophages with many non-membrane-bound lipid droplets, commonly associated with neutral lipids, were seen by TEM (Figure 6A), often in the center and at luminal surfaces of lesions. Smooth muscle cells in these lesions also contained neutral lipid droplets (Figure 6B).

View larger version (124K): [in this window] [in a new window]   Figure 6. Transmission electron microscopy (TEM) of cells from a region of yellow fluorescence by Nile red in a lesion from a rabbit sacrificed after 26 weeks on a high-cholesterol diet. (A) A macrophage with many non-membrane-bound neutral lipid droplets (l). (B) Smooth muscle cell with neutral lipid droplets (l). Bars = 1 µm. Immunohistochemical staining of Nile red yellow areas for macrophages (C) and smooth muscle cells (D). Bars = 100 µm.

Nile red and filipin fluorescence images were digitally overlaid in Figure 1E. Small patches of filipin staining are interspersed between lesions that stained with Nile red. Regions of orange Nile red staining co-localized with blue filipin staining. Most larger lesions had a "nested" appearance, with bright yellow centers surrounded by orange and blue concentric fringes. A digital overlay of the lesion edge reveals Nile red-stained cell profiles and filipin-stained fringe detail (Figure 1F). Although only relatively small FS have been illustrated, the same pattern was observed at the edges of very large FS.

	Discussion

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In this study we developed a novel fluorescence microscopy technique for in situ, semiquantitative analysis of lipid deposits in atherosclerotic aortas. The method is based on the ability of the fluorescent dyes filipin and Nile red to selectively stain non-esterified cholesterol and polar and non-polar lipids, respectively. It is useful in routinely fixed tissues and has increased sensitivity and resolution compared to most previously described methodologies. En face preparation allowed visualization of whole lesions from the smallest "pre-lesional" changes to well-developed FS that circumscribed the aorta.

Key to the validity of the lipids maps is that filipin and Nile red stain different lipid moieties specifically. Filipin is a fluorescent probe that has been used extensively to study the distribution of non-esterified cholesterol in a variety of cells and tissues (Siefert et al. 1989 ; Jaakola et al. 1988; Kruth 1985 ; Kruth and Fry 1984 ). Correlation of biochemical and fluorimetry data demonstrated a strong correlation between the intensity of staining with filipin and the content of non-esterified cholesterol in tissue, indicating that the staining technique reliably maps regions rich in non-esterified cholesterol.

Nile red has been used previously to stain neutral lipids in cells and tissues (Frank et al. 1989 ; Haban and Mesarosova 1989; Brown et al. 1988 ; Lupu et al. 1987 ; Fowler and Greenspan 1985 ). In an aqueous environment, the fluorescence of Nile red is quenched. In contrast, in a non-polar environment Nile red is dequenched and fluoresces yellow. This characteristic allows Nile red to be used as a lipid-specific fluorescent stain. In the present study, a strong correlation was shown between the intensity of Nile red yellow emission (580-596 nm) and tissue cholesteryl ester content, as determined by TLC/FID. Similar results have been reported by Koren et al. 1990 , who studied P3888D1 cells incubated with acetylated low-density lipoproteins and correlated Nile red microfluorimetry emission at 589 nm, with cholesteryl ester content measured by gas chromatography. These data indicate that the yellow emission of Nile red reliably maps regions rich in neutral lipids.

Another characteristic of Nile red is its ability to shift its fluorescence emission in response to the polarity of the lipid environment. This is in contrast to dyes such as acridine orange, for which concentration is the predominant factor (Ohkuma and Poole 1978 ). In a non-polar environment, Nile red fluoresces yellow and in a polar lipid environment the emission peak of Nile red shifts to longer wavelengths, e.g., orange, (Greenspan and Fowler 1985 ; Greenspan et al. 1985 ). This polar dependency in the Nile red emission spectra has been used to detect oxidation of LDL (Greenspan and Lou 1993 ) and to differentiate between neutral lipid and phospholipid-rich regions in cells and tissues (Klinkner et al. 1995 ; Henault and Killian 1993 ; Brown et al. 1992 ; Smyth and Wharton 1992 ; Hjelle et al. 1991 ; Bonilla and Prelle 1987 ). In the present study, although total Nile red emission intensity correlated with total non-cholesterol lipids, we were unable to directly correlate the orange emission of Nile red with phospholipid content. The lack of correlation may be due to the fluorescent quenching that accompanies the red shift in the emission spectrum of Nile red (Greenspan and Fowler 1985 ). In addition, because of a shift in the excitation maximum of Nile red, it is also possible that the filter system used in this work did not allow imaging the red-orange emission with maximal intensity. Despite the lack of correlation of fluorescence and biochemistry, TEM revealed lipid inclusions rich in phospholipids, as indicated by the presence of phospholipid whorls, in areas that were stained orange by Nile red. These data indicate that although the orange fluorescence of Nile red is not a reliable tool for quantitatively mapping phospholipids, it reliably maps regions containing a relatively polar mix of lipids.

In addition to validating the staining biochemically, we also studied the cell type and lipid inclusions responsible for the staining. TEM revealed that the smallest lesions, which stained solely with filipin, were composed of extracellular accumulations of lipid vesicles. Similar vesicles rich in non-esterified cholesterol have been described previously in the earliest atherosclerotic lesions in cholesterol-fed rabbits (Schwenke and Carew 1989b ; Mora et al. 1987 ; Kruth 1984b ) and in humans (Tirziu et al. 1995 ; Kruth 1984a ), and correspond to the areas of increased endothelial permeability (Weinbaum and Chien 1993 ) and retention of LDL (Schwenke and Carew 1989a ). Sometimes referred to as liposomes, these vesicles are believed to be derived from serum lipoproteins (Chao et al. 1994 ; Mora et al. 1987 ) and have been co-localized with apolipoprotein B in aortic lesions in rabbits (Mora et al. 1987 ) and humans (Tirziu et al. 1995 ). In the present study, TEM also revealed that the central regions of larger lesions (which stained intensely with filipin) contained aggregates of these lipid vesicles admixed with cholesterol crystals. These materials account for the very bright filipin staining seen centrally in larger FS.

TEM and IH of the margin of the lesions (which corresponded to the orange fringe) revealed that they were composed of both macrophages and smooth muscle cells that contained lipid inclusions rich in phospholipids. Similarly, TEM and IH of the centers of the larger lesions (which corresponded to the intense filipin and yellow Nile red staining) revealed that they also contained macrophages and smooth muscle cells. However, in this region the cells contained predominantly neutral lipid inclusions. Finding both macrophages and smooth muscle cells in this locations is consistent with previous results showing that early fibrous plaques in rabbits contained relatively equal numbers of smooth muscle cells and macrophages centrally (Tsukada et al. 1986 ).

In the course of our work to develop the Nile red/filipin staining technique, we conducted a preliminary evaluation of the early stages of FS formation. We found that, in general, as the FS increased in size they also increased in complexity. In addition, although the lesions were heterogeneous, the increase in complexity followed a pattern. The smallest discernible lesions (0.01-0.2 mm²) stained solely with filipin. Larger lesions (0.2-1.0 mm²) had blue (filipin) fringes with a central region that stained orange with Nile red, and the largest lesions studied (2 mm² up to FS that circumscribed the aorta) had concentric blue and orange fringes surrounding a central region that stained yellow with Nile red. This pattern, which did not follow any changes in distribution of SMC or macrophages, suggests that the complexity of the larger lesions cannot be accounted for by a simple change in cell composition. Rather, our data suggest that complexity results from changes in lipid composition, either in physical form (vesicular vs crystalline) or in chemical form (esterified vs non-esterified cholesterol, polar vs non-polar lipids) as the lesions grow in size. The change in lipid composition is probably the result of metabolic activity of macrophage and smooth muscle cell-derived foam cells: phospholipases, lipases, and acyltransferases working together to rearrange the lipid composition within a lesion (Laposata 1995 ; Aviram 1993 ; Brown and Goldstein 1983 ).

In conclusion, we have developed a new method for the investigation of the atherosclerotic lesions. En face fluorescent staining for neutral lipids, polar lipids, and non-esterified cholesterol discriminates regional differences in lipid composition of the lesions. These regional differences in lipid composition may prove more significant for understanding the evolution of a lesion than the origins of its cell composition. Further study using this dual staining method may reveal characteristic patterns of lipid deposition concurrent with the evolution and progression of atherosclerotic lesions.

	Literature Cited

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

Aviram M (1993) Modified forms of low density lipoprotein and atherosclerosis. Atherosclerosis 98:1-9[Medline]

Bonilla E, Prelle A (1987) Application of Nile blue and Nile red, two fluorescent probes, for detection of lipid droplets in human skeletal muscle. J Histochem Cytochem 35:619-621[Abstract]

Brown MS, Goldstein JL (1983) Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis. Annu Rev Biochem 52:223-261[Medline]

Brown WJ, Sullivan TR, Greenspan P (1992) Nile red staining of lysosomal phospholipid inclusions. Histochemistry 97:349-354[Medline]

Brown WJ, Warfel J, Greenspan P (1988) Use of Nile red stain in the detection of cholesteryl ester accumulation in acid lipase-deficient fibroblasts. Arch Pathol Lab Med 112:295-297[Medline]

Chao F, Blanchette-Mackie EJ, Dickens BF, Gamble W, Kruth HS (1994) Development of unesterified cholesterol-rich lipid particles in atherosclerotic lesions of WHHL and cholesterol-fed NZW rabbits. Am J Pathol 131:71-83

Folch J, Lees M, Sloane-Stanley G (1957) A simple method for the isolation and purification of total lipids from animal tissue. J Biol Chem 226:497-509[Free Full Text]

Fowler SD, Greenspan P (1985) Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections. J Histochem Cytochem 33:833-836[Abstract]

Frank HG, Gossrau R, Graf R (1989) Analysis of hepatotoxicity in maternal and fetal rats after glucocorticoid administration by lipid histochemistry and thin layer chromatography. Histochemistry 91:487-494[Medline]

Greenspan P, Fowler SD (1985) Spectrofluorometric studies of the lipid probe, Nile red. J Lipid Res 26:781-789[Abstract]

Greenspan P, Lou P (1993) Spectrofluorometric studies on Nile red treated native and oxidized low density lipoprotein. Int J Biochem 25:987-991[Medline]

Greenspan P, Mayer EP, Fowler SD (1985) Nile red: a selective fluorescent stain for intracellular lipid droplets. J Cell Biol 100:965-973[Abstract/Free Full Text]

Haban P, Mesarosva A (1989) Nile red fluorescence response from the aortic arch to serum cholesterol changes. Cor Vasa 31:419-428[Medline]

Henault MA, Killian GJ (1993) Neutral lipid droplets in bovine oviductal epithelium and lipid composition of epithelial cell homogenates. J Dairy Sci 76:691-700[Abstract/Free Full Text]

Hjelle JT, Golaiska BT, Waters DC, Steidley KR, McCarrol DR, Pavlina T, Smith SL, Lloyd JK, Dobbie JW (1991) In vivo and in vitro characterization of mesothelial lipid inclusions. Perit Dial Int 11:207-212

Jaakkola O, Kallioniemi O, Nikkari T (1988) Lipoprotein uptake in primary cell cultures of rabbit atherosclerotic lesions. A fluorescence microscopic and flow cytometric study. Atherosclerosis 69:257-268[Medline]

Klinkner AM, Waites CR, Kerns WD, Bugelski PJ (1995) Evidence of foam cell and cholesterol crystal formation in macrophages incubated with oxidized LDL by fluorescence and electron microscopy. J Histochem Cytochem 43:1071-1078[Abstract]

Koren E, Franzen J, Fugate RD, Alaupovic P (1990) Analysis of cholesterol ester accumulation in macrophages by the use of digital imaging fluorescence microscopy. Atherosclerosis 85:175-184[Medline]

Kruth HS (1985) Subendothelial accumulation of unesterified cholesterol. An early event in atherosclerotic lesion development. Atherosclerosis 57:337-341[Medline]

Kruth HS (1984a) Localization of unesterified cholesterol in human atherosclerotic lesions. Demonstration of filipin-positive, oil-red O-negative particles. Am J Pathol 114:201-208[Abstract]

Kruth HS (1984b) Filipin-positive, oil red O-negative particles in atherosclerotic lesions induced by cholesterol feeding. Lab Invest 50:87-93[Medline]

Kruth HS, Fry D (1984) Histochemical detection and differentiation of free and esterified cholesterol in swine atherosclerosis using filipin. Exp Mol Pathol 40:288-294[Medline]

Laposata M (1995) Fatty Acids. Biochemistry to clinical significance. Am J Clin Pathol 104:172-179[Medline]

Lupu F, Danaricu I, Simionescu N (1987) Development of intracellular lipid deposits in the lipid-laden cells of atherosclerotic lesions. A cytochemical and ultrastructural study. Atherosclerosis 67:127-142[Medline]

Masuda J, Ross R (1990) Atherogenesis during low level hypercholesterolemia in the nonhuman primate. I. Fatty streak formation. Arteriosclerosis 10:164-177[Abstract/Free Full Text]

Mora R, Lupu F, Simionescu N (1987) Prelesional events in atherosclerosis. Colocalization of apolipoprotein, unesterified cholesterol and extracellular phospholipid liposomes in the aorta of hyperlipidemic rabbit. Atherosclerosis 67:143-154[Medline]

Ohkuma S, Poole B (1978) Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc Natl Acad Sci USA 75:3327-3331[Abstract/Free Full Text]

Rosenfeld ME, Tsukada T, Gown AM, Ross R (1987) Fatty streak initiation in Watanabe heritable hyperlipemic and comparably hypercholesterolemic fat-fed rabbits. Arteriosclerosis 7:9-23[Abstract]

Ross R (1993) The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362:801-809[Medline]

Schwenke DC, Carew TE (1989a) Initiation of atherosclerotic lesions in cholesterol-fed rabbits. I. Focal increases in arterial LDL concentration precede development of fatty streak lesions. Arteriosclerosis 9:895-907[Abstract/Free Full Text]

Schwenke DC, Carew TE (1989b) Initiation of atherosclerotic lesions in cholesterol-fed rabbits. II. Selective retention of LDL vs. selective increases in LDL permeability in susceptible sites of arteries. Arteriosclerosis 9:908-918[Abstract/Free Full Text]

Siefert PS, Hugo F, Hansson GK, Bhakdi S (1989) Prelesional complement activation in experimental atherosclerosis. Terminal C 5b9 complement deposition coincides with cholesterol accumulation in the aortic intima of hypercholesterolemic rabbits Lab Invest 60:747-754

Smyth M, Wharton W (1992) Differentiation of A31T6 proadipocytes: a flow cytometric analysis. Exp Cell Res 199:29-38[Medline]

Tirziu D, Dobrian A, Tasca C, Simionescu M, Simionescu N (1995) Intimal thickenings of human aorta contain modified reassembled lipoproteins. Atherosclerosis 112:101-114[Medline]

Tsukada T, Rosenfeld M, Ross R, Gown AM (1986) Immunocytochemical analysis of cellular components in atherosclerotic lesions. Use of monoclonal antibodies with the Watanabe and fat-fed rabbit. Arteriosclerosis 6:601-613[Abstract]

Weinbaum S, Chien S (1993) Lipid transport aspects of atherogenesis. J Biomech Eng 115:602-610[Medline]

Williams K, Tabas I (1995) The response-to-retention hypothesis of early atherogenesis. Arterioscler Thromb 15:551-561[Free Full Text]

Wissler RW (1994) New insights into the pathogenesis of atherosclerosis as revealed by PDAY. Atherosclerosis 108:S3-S20

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