Correlation of Vasa Vasorum Neovascularization and Plaque Progression in Aortas of Apolipoprotein E−/−/Low-Density Lipoprotein−/− Double Knockout Mice
Objective— We hypothesized that apolipoprotein E (apoE)−/−/low-density lipoprotein (LDL)−/− double knockout mice might develop vasa vasorum (VV) in association with advanced lesion formation.
Methods and Results— Aortas from apoE−/−/LDL−/− mice aged 16, 18, 20, or 80 weeks were infused in situ with Microfil, harvested, and scanned with micro-computed tomography (CT). We characterized plaque volume and CT “density” as well as VV luminal volume along the aorta using Analyze 6.0 software. Results were complemented by a detailed histological plaque classification according to American Heart Association guidelines. From 16 to 80 weeks, plaque volume and VV opacified lumen volume increased with age (P<0.001). The 3-dimensional micro-CT images of arterial and venous VV trees allowed perfusion territories to be delineated. The spatial location and magnitude of VV density and adventitial inflammation were strongly correlated in advanced atherosclerotic lesions (r=0.91) and identified as an independent correlate to advanced lesions. At age 80 weeks, VV luminal volume was increased 20-fold compared with animals at age 16 weeks (P<0.001). Micro-CT showed that adventitial VV communicate with intraplaque microvessels.
Conclusion— Our results show that apoE−/−/LDL−/− double knockout mice develop VV and advanced atheromas along the aorta. Lesion volume was closely associated with amount of neovascularization in advanced atheromas.
An increasing body of experimental data suggests that vasa vasorum (VV) may play a significant role in maintaining vessel integrity and that they may contribute to the initiation and progression of different types of vascular disease of the systemic circulation.1–6
Acute and chronic inflammation of large blood vessels, as seen in atherosclerosis, is associated with proliferation of VV and is one of the predominant vascular responses that may function to sustain by perfusing the vessel wall beyond diffusion limits from the artery lumen.6 VV may also facilitate monocyte/macrophage entry into the lesion and cause intraplaque hemorrhage.7 Several observations indicate that plaque neovascularization functions to sustain the growth of subintima and media beyond the limit of diffusion from the artery lumen as suggested by the observations that VV are more prevalent as vessel wall layers increase and interruption of VV results in medial necrosis.8,9
Moreno et al10 demonstrated that neovascularization, as manifested by the localized appearance of microvessels, is increased in ruptured plaques in the human aorta. Furthermore, they could demonstrate that microvessel density is increased in lesions with inflammation, with intraplaque hemorrhage, and in thin-cap fibroatheromas. At present, ectopic neovascularization in the intima and media is considered a hallmark of advanced, vulnerable, atherosclerotic lesions.6
Detailed anatomic data about VV and plaque neovascularization are difficult to obtain because of the difficulty in their quantitative detection,) the difficulty to demonstrate their interconnectivity to other VV and the main vessel and, their very fine arborizing anatomy.
VV are generally not present in wild-type mice and they do not develop advanced atherosclerotic lesions, but apolipoprotein E (apoE)−/−/low-density lipoprotein (LDL)−/− double knockout mice develop lesions with considerable structural similarity to human atherosclerosis.12 The present study was designed to characterize atherosclerotic lesions and the localization and time course of VV neovascularization and adventitial inflammation relative to growing plaques in apoE−/−/LDL−/− double knockout mice.
Materials and Methods
Animal studies were performed according to the “German Animal-Protection Law” (1993). Approval of the institutional animal care and use committee was obtained before the start of this study.
Male apoE−/−/LDL−/− double knockout mice (n=20) receiving an atherogenic diet based on normal mouse chow but differing in total fat content (10% versus 5%), protein content (15.4% versus 22%), and cholesterol content (5% versus 0.1%). The total energy was 3990 versus 3000 kcal/kg.
Mice (Charles Rivers Wiga, Sulzbach, Germany) with an average weight of 28.5±1.8 grams were euthanized after 16 (group 1; n=8), 18 (group 2; n=5), 20 (group 3; n=5), and 80 (group 4; n=2) weeks with a fatal dose of inhaled trichlormethane. Male C57/BL mice (group 5; n=5) served as controls. The abdominal aorta was cannulated and infused with heparinized saline (10 mL of 0.9% sodium chloride with 1000 IU heparin) until the venous effluent was free of blood. A lead-containing radiopaque polymer (Microfil MV-122; Flow Tech, Carver, Mass) was infused into the aorta at a nominal pressure of 100 mm Hg. After polymerization of the compound, the heart, aorta, and lung were removed en bloc from the chest and immediately immersed in 10% neutral buffered formalin.
The aorta and lung were scanned en bloc by micro-CT systems described recently.13,14 The resulting 3-dimensional images were displayed using image analysis software (Analyze 6.0; Biomedical Imaging Resource, Mayo Clinic, Rochester, Minn). For this study, the micro-CT scanner (SkyScan1072_80kV; Artselaar, Belgium) was configured so that the side dimension of the cubic voxels was 20 μm (8-bit grayscale). Specimens in group 4 were rescanned at 20 and 5 μm at 16-bit grayscale, using a custom-built micro-CT scanner13 for more detailed analysis of VV distribution and their connectivity to atherosclerotic lesions.
Quantitative Morphometric Analysis
Atherosclerotic lesions were identified as localized thickening of the aortic wall, some with luminal encroachments. These thickenings differed in grayscale from the surrounding tissue and from the contrast-enhanced lumen.
VV and lesions were manually traced and measured on each cross-section, yielding the following parameters: lesion volume (VolL), lesion area (AreaL), lesion CT “density” (DensL), and VV luminal volume (VolVV). The volume of contrast agent (VolCA) within the lesion (an index of total VV lumenal volume) was calculated as:
where DensCA was the density of pure contrast agent over an 8-bit (255) range in which the “density” of nonopacified tissue (DensT) was measured in contrast-free, collapsed lung tissue at 8 bits (CT “density” for air=0 and DensT=75).
After micro-CT scanning, the entire tissue block was embedded in paraffin wax and sectioned. The 6-μm sections were stained with hematoxylin, eosin, and elastica and immunohistochemistry studies using anti-factor VIII-related antigen as described15 (1:1000, polyclonal rabbit anti-human; DakoCytomation) and CD11b (1:50, monoclonal anti-mouse; Pharmingen). Areas with calcified lesions were stained with 3% silver nitrate (von Kossa’s reagent) followed by a 0.1% nuclear fast red counterstaining. Goldner’s-Masson-Trichrome was used to identify collagen and smooth muscle fibers.
Movat’s pentachrome staining identified collagen and elastic fibers as well as fibrin and smooth muscle cell composition. Erythrocytes and membrane remnants were identified using antibodies against glycophorin A (CD235a, 1:100; DakoCytomation). Atherosclerotic lesions were classified as suggested by the American Heart Association.16
VV density was calculated by taking the total number of microvessels in the adventitia visualized within an histological cross-section. Monocytes within the adventitia were counted in each single slice, and the severity of inflammation was scored as 0 (no monocytes), 1 (1 to 5 cells per cross-section), 2 (6 to 25 cells), and 3 (> 25 cells) as reported previously.10
All data are present as mean±SD for all arteries. One-way ANOVA, followed by a Tukey-Kramer post hoc test with correction for multiple comparisons, was used to identify the statistical differences among groups. Individual group comparison was performed by an unpaired Student t test. Individual group comparison was performed by an unpaired Student t test. To identify independent correlates to lesion type, univariate analysis consisting of cross-tabulations of each variable was performed. A value of P<0.05 was considered significant in all analyses.
A total of 295 advanced atherosclerotic lesions were analyzed in apoE−/−/LDL−/− double knockout mice and classified according to American Heart Association guidelines.16 In our animals, 81 atheromas (type IV), 50 fibroatheromas (type Va), 54 calcified fibroatheromas (type Vb), 66 lesions with intraplaque hemorrhage (type VIb), and 44 advanced lesions with absent of lipid core (Vc) were found. No lesions with surface disruption (type VIa) or thrombosis (type VIc) were observed. Lesions type Vb and VIb were only found in animals at the age of 80 weeks.
Atherosclerotic Lesions, VV Neovascularization, and Inflammation
Images obtained by micro-CT enabled a 3-dimensional visualization of the aorta and an analysis of plaque characteristics over the entire length of the imaged sample (Figure 2). Aortic plaque extension was predominantly detected in the aortic arch after 16 weeks (Figure 2Aa). After 18, 20, and 80 weeks animals developed a greater number and more extensive lesions (P<0.001) throughout the aortic arch and the descending aorta (Table; Figure 2Bb to 2Dd). The volume of arterial and venous VV lumens showed a substantial change from 20 to 80 weeks (Table).
After 16 weeks, VV were also present in the descending aorta even in regions without atherosclerotic lesions that encroach on the aortic lumen (Figure 2).
Morphologically the lesions in the mice showed structural similarity to human atherosclerosis as they developed from early fatty lesions up to fibroatheromas and fibroproliferative calcified and complicated lesions. In animals at age 80 weeks, micro-CT showed large calcifications along the aortic arch determined by micro-CT (Figure I, available online at http://atvb.ahajournals.org). Von Kossa staining and Goldner’s-Masson-Trichrome of corresponding histological cross-sections confirmed chondrocyte-like cells and the surrounding Ca-hydroxyapatite. Total adventitial VV density was lower in fibrocalcified plaques (0.62±0.4/cross-section) when compared with other advanced lesions (P<0.0001). This was accompanied with a significant reduction of inflammatory cells in the adventitia (0.31±0.12/cross-section).
Lesions with intraplaque hemorrhage demonstrated the highest density of VV in the adventitia (13.77±1.32) and a severe adventitial inflammation (3±0). Type VIb lesions showed extensive staining for glycophorin A (Figure II, available online at http://atvb.ahajournals.org) in erythrocyte membranes colocalized with numerous cholesterol clefts, both markers of intraplaque hemorrhage (IH). In areas with IH, adventitial inflammatory cells invade the media thereby disrupting the outer elastic lamellae (Figure II).
The increasing volume of the lesions was associated with a marked increase in the appearance and volume of VV (P<0.001; Figure 3) that were predominantly localized within the adventitial compartment and at the adventitial-medial interface. There was a strong correlation between lesion CT “density” and the spatial connection of VV measured by micro-CT. Lesions with gray scale values <98 (8-bit) were not connected to VV. Quantitative micro-CT analyses demonstrated a strong linear relationship of atheromatous lesion volume to VV growth and sum of volume of microvessel lumens in the lesion (Figure 4). Logistic regression analysis revealed that VV and/or adventitial inflammation are predictive for different advanced lesion types (P<0.000). In addition, the number of VV per cross-section correlates to the number of inflammatory cells within the adventitia independent of the lesion type (r=0.91; Figure III, available online at http://atvb.ahajournals.org).
The VV could be differentiated into arterial and venous vessels by their connectivity using micro-CT (Figure 5). Moreover, at age 80 weeks, arterial VV followed a longitudinal alignment with the aortic lumen, whereas venous VV predominantly “embrace” the aorta. At age 80 weeks, advanced atherosclerotic lesions were also present on the inferior vena cava. In addition, arterial but not venous VV are present on the inferior vena cava at age 80 weeks showing a spatial distribution to the advanced atherosclerotic lesions (Figure 5). The interconnectivity of adventitial VV and plaque microvessels was demonstrated by micro-CT (Figure IV, available online at http://atvb.ahajournals.org).
The connectivity of VV indicated that venous VV drain via the intercostal veins or directly into the inferior vena cava, and that arterial VV originate from the intercostal arteries (Figure 5).
There was a significant increase in the thickness of the left ventricular wall (P<0.001) at age 20 weeks (group 3) compared with control animals at the same age (group 5) (Table).
The present study demonstrates the association among different advanced atherosclerotic lesions, adventitial VV neovascularization and adventitial inflammation in apoE−/−/LDL−/− double knockout mice. Adventitial VV density and inflammation corresponded to different lesion types and logistic regression analysis identified both variables as independent correlates to histologically different advanced atherosclerotic lesions.
Experimental and clinical evidence strongly suggests the important role of VV in vascular proliferative disorders. Consequently, VV may be suitable therapeutic targets because they may contribute to plaque development in several different ways; for example, through (1) alterations of arterial blood, oxygen, and nutrient supply to the plaque; (2) changes in venous drainage of venous VV; (3) their role as a conduit for inflammatory cells; and (4) via their influence on plaque stability and instability.
Micro-CT6,22 imaging makes it possible to quantify the number and spacing of blood vessels17 and vascular permeability,18 and to analyze changes in blood vessel walls.11 Micro-CT was shown to detect small VV that had been missed by conventional histological examination because the entire aorta can be examined at high-resolution in continuity, whereas histology sections are generally made at several-millimeter intervals along the aorta.
The association between VV and plaques is well-established, but their role as an active factor in plaque development is still indirect. Strong support for an active role is provided by Mouton et al,19 who demonstrated that inhibiting VV neovascularization is strongly associated with a reduced lesion progression in apoE-deficient mice. In that study, it was also demonstrated that inhibition of plaque angiogenesis may have beneficial effects on plaque stability. Recently, Virmani et al20 reported that a network of immature blood vessels within the plaque is a viable source of intraplaque hemorrhage, thereby providing erythrocyte-derived phospholipids and free cholesterol. The rapid change in plaque substrate caused by the excessive accumulation of erythrocytes may promote the transition from a stable to an unstable lesion.
Several studies21–26 concerning diet-induced hypercholesterolemia in pigs and its impact on VV reported an increased VV neovascularization in the adventitia layer before the development of vascular lesions. Arterial remodeling caused by hypercholesterolemia causes thickening of the vascular wall in C57/BL mice27 at age 24 weeks and resembles the structural changes present in the early phase of atherosclerotic development. However, C57/BL mice do not develop VV in the aorta. Additionally, hypercholesterolemia does not alter the delivery of oxygen to the artery wall before the formation of atherosclerotic lesions.28 Consequently, it raises the question whether hypercholesterolemia per se stimulates angiogenesis of VV. On the basis of our findings, we conclude that the increasing metabolic demand of the growing lesions does stimulate VV growth.
Coexistence of hypercholesterolemia and hypertension increased VV density in rats;29 this is of particular interest, because our animals showed severe atherosclerotic lesions in the aorta and therefore we cannot exclude hypertension because of decreased arterial compliance and/or renal artery stenosis. We did not measure the intra-arterial systemic blood pressure, but the left ventricle of the apoE−/−/LDL−/− double knockout mice showed an increase in wall thickness compared with controls. The increase in left ventricle wall thickness might be because of systemic hypertension or to the increased pulse pressure caused by stiff arteries.
There was a significant increase in the total lumen volume of VV from 16 to 80 weeks. Interestingly, at age 16 to 20 weeks, VV appeared also in regions of the descending aorta without lesions impinging on the lumen. Moreover, arterial but not venous VV are present in the wall the inferior vena cava (IVC) at age 80 weeks. The appearance of these VV is associated with the appearance of atherosclerotic lesions in the IVC. Hence, we hypothesize that whereas the growing lesions are the fundamental stimulators of VV formation, angiogenesis of the VV is necessary to support the increasing metabolic needs of the growing lesions.
In our study, immunostaining with antibody against glycophorin A revealed different stages of IH followed by the formation of buried fibrous layers within the plaque that may represent previous healed IH. Moreno et al10 demonstrated in human aortas that lesions with IH also had increased numbers of intraplaque microvessels and, in addition, microvessel density was low in lesions with mild plaque inflammation and increasingly higher in lesions with moderate or severe inflammation. The relationship between adventitial inflammation and atherosclerosis seems obvious, because in normal arteries adventitial inflammation is absent; however, once atherogenesis occurs, adventitial inflammation increases with the extent and severity of atherosclerotic plaque formation.30,31 The inflammatory reaction within the adventitia is associated with increased VV, indicating that inflammatory cells might use VV as their own means of transport into the aortic wall.
Our findings suggest a correlation of VV neovascularization and atherogenesis, but our study does not prove causality. Increase in the CT image “density” of the lesions, measured by micro-CT, could be caused by change of cellular plaque composition or caused by contrast agent within plaque microvessels, or a combination of both. Measurements of left ventricular wall thickness were performed postmortem on retrograde perfused animals. We cannot exclude that reactive constriction or varying pressures within the left chamber led to different states of dilation of the myocardium.
Summary and Conclusion
Plaque progression and VV neovascularization are seemingly inseparably linked, perhaps triggered and perpetuated by inflammatory reactions within the vascular wall. In addition to this mouse model providing a convenient method of following the progression of the atherosclerotic process, it provides potential for a convenient and rapid evaluation of methods to arrest or reverse the atherosclerotic process.
The investigation was supported in part by grants (Anschubfinanzierungsprojekt) from the faculty of human medicine of the Justus-Liebig University Giessen, Germany and National Institutes of Health grants EB000305 and HL65342 at Mayo Clinic College of Medicine. We thank G. Martels, Justus-Liebig University Giessen, Germany, and David Hansen of Mayo Clinic College of Medicine for technical assistance.
A.C.L. and R.M.B. contributed equally to the study.
- Received August 16, 2005.
- Accepted November 7, 2005.
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