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Am J Physiol Heart Circ Physiol 293: H1997-H2003, 2007. First published June 15, 2007; doi:10.1152/ajpheart.00362.2007
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INNOVATIVE METHODOLOGY

Early changes in coronary artery wall structure detected by microcomputed tomography in experimental hypercholesterolemia

Xiang-Yang Zhu,1 Michael D. Bentley,4 Alejandro R. Chade,1 Erik L. Ritman,3 Amir Lerman,2 and Lilach O. Lerman1,2

Divisions of 1Nephrology and Hypertension and 2Cardiovascular Diseases, Department of Internal Medicine, 3Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester; and 4Department of Biological Sciences, Minnesota State University, Mankato, Minnesota

Submitted 22 March 2007 ; accepted in final form 11 June 2007


   ABSTRACT
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 ABSTRACT
METHODS
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 DISCUSSION
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Changes in the structure of the artery wall commence shortly after exposure to cardiovascular risk factors, such as hypercholesterolemia (HC), but may be difficult to detect. The ability to study vascular wall structure could be helpful in evaluation of the factors that instigate atherosclerosis and its pathomechanisms. The present study tested the hypothesis that early morphological changes in coronary arteries of hypercholesterolemic (HC) pigs can be detected using the novel X-ray contrast agent OsO4 and three-dimensional micro-computed tomography (CT). Two groups of pigs were studied after they were fed a normal or an HC (2% cholesterol) diet for 12 wk. Hearts were harvested, coronary arteries were injected with 1% OsO4 solution, and cardiac samples (6-µm-thick) were scanned by micro-CT. Layers of the epicardial coronary artery wall, early lesions, and perivascular OsO4 accumulation were determined. Leakage of OsO4 from myocardial microvessels was used to assess vascular permeability, which was correlated with immunoreactivity of vascular endothelial growth factor in corresponding histological cross sections. OsO4 enhanced the visualization of coronary artery wall layers and facilitated detection of early lesions in HC in longitudinal tomographic sections of vascular segments. Increased density of perivascular OsO4 in HC was correlated with increased vascular endothelial growth factor expression and suggested increased microvascular permeability. The use of OsO4 as a contrast agent in micro-CT allows three-dimensional visualization of coronary artery wall structure, early lesion formation, and changes in vascular permeability. Therefore, this technique can be a useful tool in atherosclerosis research.

osmium tetroxide; atherosclerosis; coronary artery wall

ATHEROSCLEROSIS, the most common cause of mortality and morbidity in the Western world, is an arterial disorder characterized by thickening and loss of elasticity of the vascular wall. The disease process of atherosclerosis is initially evident mainly in the intimal layer of the artery wall, which becomes infiltrated by lipids and inflammatory cells and develops various degrees of fibrosis (15). Clinical symptoms of atherosclerosis may result from disease activity in nonobstructive lesions (18); therefore, it is important to identify and characterize early lesions. Hypercholesterolemia (HC), a major risk factor for atherosclerosis, induces lesion formation (15) and has been linked to development of coronary artery disease (1, 16). We previously showed in pigs that early lesions in diet-induced HC are characterized by increased thickness of the coronary intima (10). In more advanced lesions, accumulations of macrophages filled with numerous lipid droplets (fatty streaks) can be observed in the intima. However, studies of small early lesions may be limited, because they are difficult to detect and characterize by two-dimensional techniques, such as angiography and conventional histology, without destruction of the vessel.

In addition to intimal proliferation, early vascular changes in HC are characterized by increased permeability, suggesting loss of vascular integrity and the barrier function of the endothelium. This increased permeability may result from inflammatory activity in the vascular wall, which may upregulate expression of the vascular endothelial growth factor (VEGF) (19). We previously showed increased vascular permeability in hypercholesterolemic (HC) pigs in vivo in response to cardiac challenge induced by intravenous adenosine (13). However, because of inadequate spatial resolution and sensitivity of in vivo imaging techniques, it remains unclear whether these seemingly transient changes in vascular permeability are dynamic or structural and can also be detected during basal conditions.

Micro-computed tomography (CT) is a powerful experimental imaging technique that is able to generate continuous thin-section images of small specimens. The technique has been used successfully for visualization of the lumens and branching patterns of the myocardial (25, 26) and renal (24) vasculature, but only structures with different attenuation values can be distinguished and quantified (11). The contrast agent often used in micro-CT scanning, Microfil, is an intravascular agent that illustrates the lumen and three-dimensional architecture of the vasculature, but not the structure of the vessel wall, which lacks contrast. OsO4 is a contrast agent that specifically interacts with lipids (22) and, under certain circumstances, with proteins as well (12). Although conventionally used for electron microscopy, OsO4 has been shown to be detectable by X-rays (2, 20), so it may allow characterization of the vessel wall and surrounding tissues during micro-CT scanning. Furthermore, the leakage of OsO4 out of the blood vessels may also be useful for assessment of vascular permeability.

Thus the purpose of the present study was to evaluate the feasibility of using OsO4 in micro-CT scanning for characterization of early changes in the coronary artery wall during early atherosclerosis.


   METHODS
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 METHODS
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All procedures using animals were approved by the Institutional Animal Care and Use Committee. Two groups of age- and body weight-matched female domestic pigs were studied after they were fed a normal (n = 9) or an HC (n = 9) diet (2% cholesterol and 15% lard by weight; TD93296, Harlan Teklad, Madison, WI) for 12 wk (13, 26). The plasma lipid profile (Roche, Nutley, NJ) was determined after 12 wk of diet in both groups, and mean arterial pressure was obtained by an arterial catheter. After euthanasia by pentobarbital sodium infusion (100 mg/kg iv; Sleepaway, Fort Dodge Laboratory, IA), the heart was removed. A piece of the left ventricular wall was sectioned and kept at –80°C for in vitro studies. The left anterior descending coronary artery (LAD, 5 animals in each group) or the right coronary artery (4 animals in each group) was cannulated and infused with 0.9% saline-heparin (to flush out blood) at a physiological pressure closely matched to the pig's blood pressure (flow rate = 0.9 ml/min) (14, 26) and, subsequently, with formaldehyde. For comparison, the LAD of an additional normal pig was similarly infused with an intravascular silicone rubber (Microfil, MV-122, Flow Tech). The heart was then immersed in formaldehyde for in vitro studies.

Micro-CT procedure. After unbound formaldehyde was flushed from the vasculature by infusion of the coronary arteries with 60 ml of 0.9% saline, 10 ml of 1% OsO4 solution were hand injected in 1-ml increments into each artery every 6 min for 1 h. A portion of the distal epicardial coronary artery, including a piece of the attached ventricular myocardium (~2 x 1 x 1 cm), was then sectioned, prepared, and scanned by micro-CT, as previously described (25, 26). One Microfil-filled coronary artery was scanned at the Brookhaven National Laboratory. Images were digitized for reconstruction of three-dimensional volume images, which consisted of cubic voxels of 6 or 20 µm on a side, and were displayed at 12- or 40-µm voxels, respectively, for subsequent analysis.

Images were analyzed with the ANALYZE software package (Biomedical Imaging Resource, Mayo Clinic, Rochester, MN). Images (composed of ~500 slices each) were displayed cross sectionally and longitudinally, and the coronary arteries were examined for the presence of lesions. In addition, permeation of OsO4 into the vessel wall allowed detection of three layers of different densities in the coronary artery wall. The middle layer, which had higher density, corresponded to the media (Fig. 1). This layer and the arterial lumen were traced in each cross section within 1 cm of the artery, and their volumes and the media-to-lumen volume ratio were calculated.


Figure 1
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  		Fig. 1. A: micro-computed tomographic (CT) image of a normal coronary artery showing partial lumen (L), intima (dashed arrow, a), media (b), adventitia (c), perivascular connective tissue (d), and myocardium (e). B: corresponding histological section of coronary artery wall in A showing similar vessel wall structure. Solid arrows show spatially similar OsO4 accumulation. C: micro-CT image of a normal coronary artery obtained using an intravascular contrast agent (Microfil). Vascular wall layers could not be identified. Arrows show vessel wall vasa vasorum.

 
Microvascular permeability to OsO4 was assessed in <500-µm-diameter microvessels. The area of the opacified region surrounding each microvessel was measured and normalized to vessel area. Similarly, the area enclosing perivascular OsO4 around each epicardial coronary artery was measured and normalized to lumen area. In addition, for standardization of OsO4 attenuation values, the average opacity of a 40 x 60 pixel region of interest was measured in each sample background (embedding material) and subtracted from the corresponding OsO4 opacity.

Histology. For evaluation of perivascular and myocardial lipid deposition, 5-µm frozen sections were cut from left ventricular samples of normal and HC animals and stained with Oil Red O (Sigma, St. Louis, MO). In addition, after micro-CT scanning, the scanned samples (including myocardium and epicardial coronary artery) were unpacked from the scanning tube and embedded in paraffin, and 5-µm-thick sections were cut from each block and stained with hematoxylin and eosin or trichrome. For evaluation of the spatial relationship of myocardial OsO4 distribution to microvascular permeability, slices (1 from each pig) were also immunostained with antibody against VEGF (1:50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) according to a standard immunohistochemical protocol (5, 14). Samples in which early lesions were identified in micro-CT were sliced at the approximate levels of the lesion, with anatomic landmarks identified during micro-CT image analysis used for guidance.

Cross sections of the myocardium in the isolated sample (1 per animal) were examined as we previously described (25).

Statistical analysis. Continuous data are expressed as means ± SE, and unpaired t-test was applied to compare the difference between two groups. Statistical significance was accepted if P ≤ 0.05.


   RESULTS
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Total and low-density lipoprotein cholesterol levels were significantly higher in HC than in normal pigs, but triglyceride levels were similar between the groups. Body weight and blood pressure were similar between the groups (Table 1).


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  		Table 1. Systemic and coronary wall data obtained using OsO4 staining and micro-CT scanning in normal and HC pigs

 
OsO4 enhances visualization of vessel wall structure. The vascular lumen, media, adventitia, and perivascular tissue could be identified in micro-CT images by differential densities due to OsO4 permeation and distribution. In the normal epicardial coronary artery, a thin and faint intimal layer could be identified in micro-CT images (Fig. 1A). OsO4 density in the middle layer was higher, probably because of its binding to a greater number of smooth muscle cells, and corresponded to the media. The highest density of OsO4 appeared in the adventitial layer (Fig. 1A). The micro-CT images were comparable to those obtained in histological sections (Fig. 1B). These structures could not be identified separately in micro-CT images with the standard intravascular contrast Microfil, which highlighted only the lumen (Fig. 1C).

OsO4 distribution in the media and adventitia of the epicardial coronary artery wall was similar in normal and HC arteries (Fig. 2, A and B). There was no OsO4 accumulation in the intima of normal pigs (Fig. 2A). However, in two of nine HC pigs, early lesions could be detected by examination of longitudinal tomographic displays of a vessel segment of the epicardial coronary arteries. Very early changes could be identified by increased subintimal accumulation of OsO4 without encroachment on the lumen (Fig. 2B). Slightly more advanced early lesions were characterized by mild nonobstructive protuberance into the lumen and increased local density of OsO4, likely indicating increased local permeability and intimal lipid accumulation (Fig. 2C). The three-dimensional tomographic sections allowed examination of the extent of the lesion along the vessel and at different angles (cross-sectional or longitudinal; Fig. 2). These lesions were not detected in randomly selected histological sections. Similarly, measurements of medial and luminal volumes enabled calculation of three-dimensional media-to-lumen ratio, although it was not significantly different between normal and HC animals (Table 1).


Figure 2
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  		Fig. 2. Longitudinal (top) and cross-sectional (bottom) views of coronary arteries obtained from normal (A) and hypercholesterolemic (HC, B and C) pigs show very early changes of increased subintimal accumulation of OsO4 without encroachment on the lumen (B). Slightly more advanced early lesions were characterized by mild nonobstructive protuberance into the lumen and increased local density of OsO4 (C).

 
Vascular permeability. In epicardial coronary arteries, the area of perivascular OsO4 in HC animals was not different from that in normal animals, but its opacity was higher in HC (Table 1), indicating increasing retention of OsO4 in this area. Furthermore, strikingly more extensive OsO4 permeation and binding around microvessels was observed in sections from HC than normal pigs (Fig. 3, Table 1), indicating increased microvascular permeability. Interestingly, subsequent immunohistochemistry of the same sections indicated that increased microvascular permeability in HC was spatially correlated with increased local VEGF expression (Fig. 4), supporting the relationship between microvascular perivascular OsO4 leakage and microvascular permeability. Oil Red O-stained myocardium showed greater lipid accumulation in the adventitia and in some myocytes surrounding the epicardial coronary arteries in HC pigs, but no staining was evident around intramyocardial microvessels (Fig. 5). Trichrome-stained samples showed accumulation of OsO4 aggregates in fat cells in the adventitia, especially in areas with abundant vasa vasorum (Fig. 5).


Figure 3
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  		Fig. 3. Representative micro-CT images of myocardial microvessels from normal and HC pigs. More extensive opacification surrounding microvessels in HC suggests increased microvascular permeability.

 

Figure 4
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  		Fig. 4. Representative myocardial immunohistochemistry of vascular endothelial growth factor (VEGF, left) and corresponding micro-CT image (right) of the same section. Note similar distribution of VEGF (red) and perivascular OsO4 (white). Perivascular distribution of VEGF and OsO4 was increased in HC compared with normal pigs. Note similar distribution of VEGF (red, black arrow) and perivascular OsO4 (white, white arrow).

 

Figure 5
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  		Fig. 5. Representative histological sections from the heart of an HC pig stained with trichrome (A and B) and Oil Red O (C and D). Trichrome staining shows brown OsO4 aggregated in fat cells adjacent to the vasa vasorum (B, arrow). Lipid staining (arrow, Oil Red O) was evident in the perivascular region of a larger coronary artery, but not around intramyocardial microvessels (D, arrow).

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
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The present study has demonstrated that OsO4 in conjunction with micro-CT scanning enhances the visualization of the coronary artery wall structure and allows novel experimental assessments of early lesion formation and vascular permeability in HC pigs.

OsO4 has been used in tissue fixation for more than a century. When tissue is fixed by OsO4, it becomes bound by many lipid-abundant structures, such as membranes, mitochondria, nucleoli, Golgi bodies, and lipid droplets (23). Since OsO4 strongly scatters electrons, it constitutes an excellent contrast for electron-microscopic studies (21). Importantly, all the OsO4 taken up by fresh tissues is bound by unsaturated lipids (6), whereas in tissues treated with acid fixatives, denatured proteins react strongly with OsO4, which then becomes a protein stain (12). In the present study, OsO4 injected through coronary arteries previously fixed by formaldehyde may have stained not only lipids but, also, proteins. For this reason, OsO4 was useful for visualization of some of the vessel wall structure, since it distributed in the vessel wall and myocardium with different densities in micro-CT images. The thin intimal layer was difficult to identify in micro-CT images of the normal epicardial coronary artery, whereas intimal thickening (and, possibly, lipid accumulation) could be clearly identified in HC pigs. In the middle layer, which corresponded to the media, as shown histologically, OsO4 density was increased; i.e., it was probably bound to the smooth muscle cells abundant in the media. The spotty distribution of OsO4 in the medial layer of HC pigs might be related to binding to a heterogeneous smooth muscle cell population (7). The highest density of OsO4 in epicardial vessels appeared in a patchy distribution in the periphery of the adventitial layer. This may have reflected OsO4 that reached this region through the vasa vasorum and then bound to adjacent fat cells aggregated in this region (Figs. 1B and 5), since, as a heavy (~254-Da) metal, OsO4 has limited penetration ability (3). This notion is further supported by the comparably patchy distribution pattern of the vasa vasorum observed in the LAD, which shows low-density vasa vasorum territories, especially on the myocardial aspect of the coronary artery wall (9). Furthermore, this may have also contributed to the spotty pattern observed in the media.

In contrast to epicardial coronary arteries, OsO4 could penetrate the thin microvascular wall and diffuse into perivascular tissue. In fact, the degree of leakage of OsO4 into the perivascular and extravascular compartment may have further physiological significance. Changes in endothelial permeability are major common pathogenic mechanisms in atherosclerosis. Our previous studies showed that HC elicits vascular endothelial dysfunction and increases vascular permeability in vivo (13). However, it remained unclear whether this was a transient, reversible phenomenon associated with increased cardiac demand or a structural abnormality. By use of micro-CT scanning with OsO4 as a contrast agent, the present study demonstrated that it is likely to be structural, because the increased permeability is observed ex vivo. Furthermore, the present study suggests that the increased permeability is localized to intramyocardial microvessels and likely due to increased VEGF expression. VEGF is a major growth factor that increases vascular permeability, induces angiogenesis, and may also disrupt vascular barrier function in diseased tissues (19). We previously showed that its myocardial (26) and renal (4) expression increases in HC, likely due to proinflammatory mechanisms. By uncoupling endothelial cell-cell junctions, VEGF can then increase vascular permeability and edema, which may contribute to inflammatory cell infiltration and adhesion. Our study suggests that VEGF may indeed play such a role in the myocardium in HC. In contrast to the epicardial coronary artery, in which increased perivascular OsO4 density in HC was associated with greater lipid accumulation surrounding vessels in HC pigs, increased OsO4 permeation surrounding intramyocardial microvessels was not accompanied by local Oil Red O staining or lipid binding, suggesting that it resulted from increased microvascular permeability in HC pigs.

Langheinrich et al. (11) demonstrated the feasibility of analyzing the morphology of advanced coronary artery plaques with micro-CT without the use of contrast media. Although this approach is very useful, its value in visualization and differentiation of structures with similar densities, such as vessel wall layers, is limited (11). Indeed, OsO4 significantly enhances the contrast of the coronary artery wall layers and myocardial structure and provides images that resemble those observed histologically. Therefore, it allowed for measurement of the medial and luminal volumes and calculation of three-dimensional media-to-lumen ratio. In contrast to hypertensive animals (25), this ratio was not significantly different between normal and HC animals.

Compared with conventional histological analysis, the use of micro-CT for assessment of vascular wall structure may offer several advantages. With a relatively short acquisition time (11), micro-CT provides fast analysis of an entire coronary segment. Because it permits visualization of continuous sections of a vascular segment, micro-CT might be useful for detection of early and small atherosclerotic lesions ex vivo in the coronary vessels that may be difficult to detect using conventional histological or even micro-CT analysis. Furthermore, micro-CT may enable characterization of lesion morphology. Acquisition and reconstruction of cross sections and longitudinal three-dimensional images of the vessels and lesions allow determination of the three-dimensional structure, volume, and plaque burden of the lesions or other vascular structures. This feature may be useful for the investigation of the progression of atherosclerosis. As an ex vivo but nondestructive approach, further histological analysis can be performed in the same specimens, such as the VEGF immunostaining performed in the present study. In contrast to histology, micro-CT allows consecutive slice-to-slice registration with minimal distortion. The use of micro-CT may therefore be of great advantage in ex vivo clinical or experimental studies.

In the present study, we used the LAD and right coronary artery, which have similar arterial wall structures (8). Since we tested the feasibility of using OsO4 in micro-CT analysis only in early lesions, the benefit of this technique in analysis of the structure of advanced lesions remains to be determined. However, amplified contrast enhancement may also help characterize more advanced lesions. The toxicity (17) of OsO4 and its restriction to ex vivo techniques also limit its application.

In summary, our study demonstrates that OsO4 provides sufficient contrast for visualization of coronary wall structure and affords three-dimensional analysis of the structure, permeability, and lesion formation in the vessel wall. In larger coronary arteries, OsO4 accumulation may be affected by greater adventitial lipid deposition in HC. The use of micro-CT may be beneficial in ex vivo clinical or experimental studies of atherosclerosis.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
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 GRANTS
REFERENCES
 
This study was partly supported by National Institutes of Health Grants DK-73608, HL-77131, and HL-65342.


   ACKNOWLEDGMENTS
 
The authors acknowledge the image acquisition carried out at the National Synchrotron Light Source, Brookhaven National Laboratory, which is supported by the US Department of Energy, Division of Material Sciences and Division of Chemical Sciences, under Contract DE-AC02-98CH10886. Present address of A. R. Chade: Dept. of Physiology and Biophysics, Univ. of Mississippi Medical Ctr., 2500 N. State St., Jackson, MS 39216-4505.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. O. Lerman, Div. of Nephrology and Hypertension, Mayo Clinic College of Medicine, 200 First St., SW, Rochester, MN 55905 (e-mail: lerman.lilach{at}mayo.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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