Increased Neovascularization in Advanced Lipid-Rich Atherosclerotic Lesions Detected by Gadofluorine-M–Enhanced MRI

Abstract

Background

Inflammation and neovascularization may play a significant role in atherosclerotic plaque progression and rupture. We evaluated gadofluorine-M–enhanced MRI for detection of plaque inflammation and neovascularization in an animal model of atherosclerosis.

Methods and Results

Sixteen rabbits with aortic plaque and 6 normal control rabbits underwent gadofluorine-M–enhanced MRI. Eight rabbits had advanced atherosclerotic lesions, whereas the remaining 8 had early lesions. Magnetic resonance atherosclerotic plaque enhancement was meticulously compared with plaque inflammation and neovessel density as assessed by histopathology. Advanced plaques and early atheroma were enhanced after gadofluorine-M injection. Control animals displayed no enhancement. After accounting for the within-animal correlation of observations, mean contrast-to-noise ratio was significantly higher in advanced plaques than compared with early atheroma (4.29±0.21 versus 3.00±0.32; P=0.004). Macrophage density was higher in advanced plaques in comparison to early atheroma (geometric mean=0.50 [95% CI, 0.19 to 1.03] versus 0.25 [0.07 to 0.42]; P=0.05). Furthermore, higher neovessel density was observed in advanced plaques (1.83 [95% CI, 1.51 to 2.21] versus 1.29 [0.99 to 1.69]; P=0.05). The plaque accumulation of gadofluorine-M correlated with increased neovessel density as shown by linear regression analysis (r=0.67; P<0.001). Confocal and fluorescence microscopy revealed colocalization of gadofluorine-M with plaque areas containing a high density of neovessels.

Conclusion

Gadofluorine-M–enhanced MRI is effective for in vivo detection of atherosclerotic plaque inflammation and neovascularization in an animal model of atherosclerosis. These findings suggest that gadofluorine-M enhancement reflects the presence of high-risk plaque features believed to be associated with plaque rupture. Gadofluorine-M plaque enhancement may therefore provide functional assessment of atherosclerotic plaque in vivo.

There is greatly increased awareness of the vital role that inflammation and neovascularization play in the natural history of atherosclerosis and in potentiation of atherosclerotic plaque rupture. Although the role of inflammation and macrophage infiltration has been widely investigated,^1,2 the recent involvement of neovascularization as an independent predictor for plaque rupture has led to an increased interest in this area.^3,4 Neovessel formation originating from the vasa vasorum is implicated in intraplaque hemorrhage, which dramatically increases inflammation and thereby augments the risk of plaque rupture.⁵

Plaque neovessels have been difficult to detect in vivo until recently.⁶ High-resolution MRI allows for accurate quantification of plaque components in vivo. Moreover, contrast-enhanced MRI has been shown to improve plaque characterization.^7,8

Gadofluorine-M represents a new class of contrast agents successfully used by our group⁹ and others¹⁰ for plaque detection and characterization in vivo. Gadofluorine-M behaves in vivo as a blood pool agent as the result of its inherent properties. It tends to persist longer in small vascular structures such as neovessels because it has a long half-life in blood. We designed the present study to test the hypothesis that atherosclerotic plaque enhancement is related in part to inflammation and neovascularization. Therefore, we evaluated the feasibility of using gadofluorine-M–enhanced MRI in vivo to assess macrophage and neovessel content in atherosclerosis. In addition, we assessed colocalization of gadofluorine-M and neovessels within atherosclerotic plaques using laser-scanning confocal microscopy.

Methods

Animal Protocol

Aortic atherosclerotic lesions were induced in New Zealand White rabbits (n=16; age, 3 months; body weight, 3.0 to 3.5 kg; Covance, Princeton, NJ) under anesthesia (intramuscular Ketamine, 35 mg/kg, and Xylazine, 7 mg/kg) by a single aortic denudation as previously described.⁹ Rabbits were fed a high-cholesterol diet (Purina rabbit chow, 0.2% cholesterol; Research Diets, New Brunswick, NJ) for a minimum of 2 months and subsequently were divided into 2 groups. The first group (Ea) was fed a high-cholesterol diet for a total duration of 2 months and the second group (Ad) was fed a high-cholesterol diet for a minimum of 8 months. Animals underwent MRI at 2 months (range, 2 to 2.5 months for the Ea group; n=8), and 8 months (range, 8 to 9 months for the Ad group; n=8) after balloon injury. Animals were euthanized at these time points for validation studies (described below) and for a separate analysis of histopathology end points. The Mount Sinai Institute of Animal Care and Use Committee approved all experiments. Six normal New Zealand White rabbits were used as control animals. Balloon injury was not performed on the control animals, and they were not fed a hypercholesterolemic diet.

MRI

The MRI protocol used was based on previously validated work.⁹ Rabbits were sedated with Ketamine/Xylazine (as above) and imaged supine in a 1.5-T MRI system (Sonata, Siemens, Germany). Transverse images of the abdominal aorta were obtained using a T₁-weighted 2D segmented gradient-echo sequence with a combination of an inversion-recovery and diffusion-based flow suppression preparatory pulse as reported by our group recently.⁹ Images were obtained before (precontrast) and 24 hours after intravenous 50 μmol/kg body weight gadofluorine-M injection (postcontrast) in both groups (Ea and Ad). The MR parameters were as follows: repetition time, 300 ms; echo time, 4 ms; inversion time, 220 ms; flip angle, 20°; and spatial resolution, 0.4×0.4×2 mm³.

Contrast Agent

Gadofluorine-M (Bayer Schering Pharma AG, Berlin, Germany), is highly water-soluble and an amphiphilic gadolinium (Gd)-based contrast agent. It has a low molecular weight (1530 Da) with a macrocyclic Gd chelate complex (Gd-DO3A derivative) as well as a perfluorinated side chain (perfluoroalkyl tail). Due to the hydrophobic character of the fluorinated side chain, gadofluorine-M assembles like small aggregates or micelles in diluted solution. Because of the long plasma half-life in rabbits (≈10 hours),⁹ gadofluorine-M behaves like a blood pool agent. Properties of the compound have been reported elsewhere.^11,12 Carbocyanine-labeled gadofluorine M was used for colocalization with neovessel staining using laser-scanning confocal microscopy. The excitation and emission maxima of this compound are 581 nm and 596 nm, respectively.¹³ Gadofluorine-M and Carbocyanine-labeled gadofluorine-M form mixed micelles in water.

Histopathologic Analysis and Assessment of Gadofluorine-M Deposition

Histopathologic analysis was systematically performed for validation of MRI measurements with histomorphometry. An experienced pathologist (K.R.P.) blinded to the MR findings performed the analysis, using the classification from the Committee on Vascular Lesions of the Council of Atherosclerosis, American Heart Association.²

Rabbits were euthanized, after acquisition of the last set of MR images, by intravenous injection of sodium pentobarbital (120 mg/kg) and heparin (1000 U/kg) to prevent postmortem thrombosis. The animals in each group were randomly chosen for (1) paraformaldehyde tissue fixation (4 in the Ea group and 4 in the Ad group), or (2) frozen tissue fixation (4 in the Ea group and 4 in the Ad group). Coregistration was performed carefully by using the position of the renal arteries and iliac bifurcation.

The aortas were excised and transferred in 2% paraformaldehyde and perfusion-fixed. Serial sections of the aorta were cut at 3-mm intervals matching the corresponding MR images. The selected aortic specimens were embedded in paraffin, sectioned 5 μm in thickness, and stained with hematoxylin and eosin as well as Masson trichrome elastin stain for American Heart Association plaque classification.²

The aortas were excised and tissue specimens were cryoprotected with 30% sucrose and frozen in OCT (Tissue-Tek Optimal Cutting Temperature, Sakura Finetek Inc, Torrance, Calif) and stored at −80°C. Thereafter, 8-μm-thick sections were analyzed for the presence of Carbocyanine-labeled gadofluorine-M by red fluorescence on a Zeiss Axiophot microscope (Zeiss, Thornwood, NY).

Sections were additionally stained by immunohistochemistry for macrophage detection (RAM11, Dako Inc, 1:200) and microvascular endothelium or neovascularization (vasa vasorum derived) (CD31-M0823-Dako Inc, 1:30). Specificity of antibodies was confirmed by routine positive and negative controls running in parallel for each batch of staining experiments. Appropriate controls, including unstained tissue sections to detect autofluorescence, were also examined with laser-scanning confocal microscopy.

Image Analysis

MR Images

MR images were analyzed on a dedicated workstation (Leonardo, Siemens, Germany). Wall and lumen signal intensities (SIs) were determined using standard region-of-interest measurements on the corresponding MR images.⁹ The normalized contrast-to-noise ratio (CNR), CNR=SI_wall−SI_lumen/SD_noise, was calculated as previously described.⁹ An experienced observer drew all regions of interest. CNR was calculated before and after (24 hours) gadofluorine-M injection in all groups (Ea, Ad, and control groups). The standardized protocol ensured identical slice position for the precontrast and postcontrast images.

Histopathology

Inflammatory cells were identified in a high-power field with a ×40 magnification objective and defined as RAM11/CD3-positive mononuclear round cells. Macrophage density (macrophage area divided by plaque area) was also reported. Plaque neovessels were defined as tubuloluminal CD31-positive capillaries recognized in cross-sectional and longitudinal profiles as identified by immunohistochemistry in the intima, in the media and in the adventitia at a ×40 magnification objective. Neovessel density was calculated by dividing the total number of microvessels by plaque area (mm²). Quantification was regionally tabulated for 2 contiguous, nonoverlapping, transmural sites for each individual section. Cross-sectional plaque areas were manually traced on each aortic section using ImagePro Plus (Media Cybernetics).

Statistical Analysis

Continuous data were assessed for normality using normal plots. CNR followed a gaussian distribution, and data are expressed as mean±standard error. As neovessel and macrophage density followed a nongaussian distribution, these variables were log-transformed and the geometric mean and 95% CI are presented. For all analyses, standard errors were calculated using Huber-White sandwich estimators. This approach takes into account the within-rabbit correlation of data. Comparisons of CNR and log-transformed neovessel and macrophage density between the Ea and Ad groups were performed using general linear models, accounting for within-rabbit correlation of data. Correlations were established between CNR and plaque area, neovessel density, and macrophage density using Pearson correlation coefficient and linear regression analysis. The SPSS 16.0 and Stata 10.0 software were used for the analysis.

Results

All MR images (n=110) were interpretable. All post–gadofluorine-M injection images showed enhancement, and aortic atherosclerotic lesions were readily detected in both atherosclerotic groups of rabbits (advanced and early atherosclerotic groups), as demonstrated by Figure 1. The average CNR measured in the advanced atherosclerotic rabbit group (n=8 rabbits) was significantly higher than the average CNR measured in the early atherosclerotic group (n=8, 4.29±0.21 versus 3.00±0.32, respectively; P=0.004). Post–gadofluorine-M injection MR imaging showed no enhancement of the abdominal aortas in all control animals (n=6).

Figure 1.

Transverse T₁-weighted MR images of atherosclerotic rabbit abdominal aorta at 2 different time points: A, Precontrast imaging; B, 24 hours after gadofluorine-M injection. C, Corresponding histopathologic section is stained with combined Masson elastin trichrome. The atherosclerotic plaque is rich in lipids (3 to 9 o’clock positions), matching the plaque enhancement seen with gadofluorine-M. Magnification ×4. Ad indicates adventitia; L, lumen; NC, necrotic-core.

Histopathologic analysis revealed the presence of neovessels within the adventitia, the media, and more interestingly, within the intima as illustrated by Figure 2. Tubuloluminal CD31-positive capillaries were identified by immunohistochemistry and visually recognized in cross-sectional sections. As assessed by histopathology, neovessel density was higher in advanced plaques when compared with early atheroma (geometric mean=1.83 [95% CI, 1.51 to 2.21] versus 1.29 [0.99 to 1.69]; P=0.050), as shown in Figure 3. Furthermore, macrophage density was higher in advanced plaques (geometric mean=0.50 [0.19 to 1.03] versus 0.25 [0.07 to 0.42]; P=0.05).

Figure 2.

Histological example of an atherosclerotic rabbit aorta stained for neovessels. Medium-power images (×20) of microvessels at atherosclerotic plaque intima (A), media (B), and adventitia (C), detected with monoclonal endothelial cell marker CD31 (a unique marker). High-power images (×40) of the intima (D), media (E), and adventitia (F) of the atherosclerotic plaque demonstrate the tubuloluminal CD31-positive capillaries identified in cross sections as atherosclerotic neovessels.

Figure 3.

Diagram depicts (upper panel) contrast-to-noise ratio (CNR). Neovessel density (middle panel); macrophage density (lower panel); for early (Early) atherosclerotic rabbit group (black bars) and advanced (Advanced) atherosclerotic rabbit group (white bars). *P<0.05.

Confocal and fluorescence microscopy revealed colocalization of gadofluorine-M with plaque areas having a high neovessel and macrophage density, as demonstrated by Figure 4.

Figure 4.

Advanced atherosclerotic plaques from the same aortic specimen are shown in both rows using high-power magnification (×40) and using confocal microscopy (A), binocular microscopy with combined Masson elastin trichrome (B), CD-31 staining (C) for neovessel detection, and RAM-11 staining (D) for monocyte/macrophage detection. Carbocyanine-labeled gadofluorine-M is seen as red within the atherosclerotic plaque (A). The combined Masson elastin trichrome (B) shows the lipid-rich area within the plaque corresponding to the neovessel formation (C) and to the accumulation of monocytes/macrophages in brown (D) in the matching area of gadofluorine-M accumulation (A).

Using linear regression analysis, there was a significant correlation (r=0.67; P<0.001) between neovessel density and gadofluorine-M plaque enhancement (Figure 5). The correlation between CNR and plaque area was not statistically significant (r=0.17; P=0.064).

Figure 5.

Diagram depicts Pearson correlations between contrast-to-noise ratio (CNR) and neovessel density (upper panel), atherosclerotic plaque area (middle panel), and macrophage density (lower panel). A good correlation was found between CNR and neovessel density (r=0.67; P<0.001).

Discussion

In humans, the vasa vasorum is present in most major arteries, including the aorta, coronary, carotid, and femoral arteries.¹⁴ Pathologic neovascularization of the vessel wall is a consistent feature of atherosclerotic plaque development and progression.^15,16 Microvessels or neovessels are increased in coronary lesions from patients with acute myocardial infarction, suggesting a potential role for neovessels in plaque rupture.¹⁷ Plaque neovessels are often found in plaque areas rich in macrophages and T cells, which can activate lymphocyte-induced angiogenesis.¹⁸ Their close proximity to inflammatory cells and their expression of endothelium adhesion molecules (such as vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin) both suggest that neovessels may recruit inflammatory cells into atherosclerotic plaques contributing further to plaque vulnerability.¹⁹ Neovessels have been demonstrated recently as a source of intraplaque hemorrhage.²⁰ Neovessel-related intraplaque hemorrhage has been associated with lipid-core expansion.²¹ Furthermore, intraplaque hemorrhage is a potent stimulus for macrophage activation and foam cell formation, thereby increasing plaque inflammation.²² Neovessels also play a role in plaque hemorrhage associated with the development of symptoms in cerebrovascular disease.²³ Moreover, angiogenesis occurs in association with remodeling and protease activation in the surrounding tissues.²⁴ Therefore, factors that stimulate plaque angiogenesis could also contribute to processes that promote plaque disruption.

In this study, we demonstrated the ability of gadofluorine-M–enhanced MRI to detect atherosclerotic plaques with features of vulnerable or high-risk plaques. We established a good correlation between gadofluorine-M–mediated atherosclerotic plaque enhancement and neovessel density of the plaques. Furthermore, we have direct evidence of gadofluorine-M colocalization with neovessel-rich plaque areas using confocal and fluorescence microscopy. These data suggest that plaque enhancement is in part mediated through neovasculature of atherosclerotic plaques. In addition, no correlation was found between plaque volume and plaque enhancement. These data are consistent with the findings using contrast-enhanced MRI for the assessment of tumor neovascularization, showing no correlation between tumor size and neovessel density.^25–27

Several MRI techniques have been applied to the quantification and measurement of neovessels in vivo.^25,26 In one study of atherosclerosis, contrast-enhanced MRI performed on pigs showed an enhancing outer rim surrounding carotid artery walls.²⁸ Similar results have been demonstrated in humans.²⁹ The cause of outer rim enhancement was thought to be increased vascularity of the adventitial vasa vasorum feeding the plaque neovasculature. Using dynamic contrast-enhanced MRI, recent investigations of atherosclerosis imaging demonstrated the feasibility of this technique to measure and quantify the extent of plaque neovascularization.^30,31 Molecular MRI has also been successfully applied for imaging atherosclerosis by the use of a specific contrast agent targeting neovascularization with a nanoparticle targeted to αvβ3-integrins (a neovascularization-specific target).⁶

Gadofluorine-M behaves in vivo as a blood pool agent as the result of its inherent properties and long plasma half-life. Blood pool agents (ie, purely intravascular contrast agents) remain in the blood for a prolonged time compared with conventional contrast agents, which diffuse quickly into the interstitial space.³² Gadofluorine-M tends to persist longer in small vascular structures such as neovessels because it has a long half-life in blood. Microvessels derived from the vasa vasorum have increased vascular permeability³³ and promote exchange between atherosclerotic plaques and the blood pool. Some investigations have demonstrated that apolipoproteins A-I and B were observed in proximity to neovessels, suggesting local lipid deposition via adjacent microvasculature.^15,34 This is consistent with our previous findings suggesting that gadofluorine-M tends to accumulate in proximity to lipid-rich areas of atherosclerotic plaques.⁹

The clinical importance of plaque neovascularization is suggested by studies that show a higher prevalence of neovascularization in lesions with plaque rupture, intraplaque hemorrhage, or unstable angina.^{3,5,17,20,35,36} The methodology described is this manuscript is currently applicable in human without any major changes. The present limitation is represented by compound safety issues for human use. However, a noninvasive imaging technique that is able to reliably detect the degree of atherosclerotic plaque neovascularization in addition to assessing plaque composition and inflammation would greatly enhance our ability to risk-stratify patients and identify vulnerable or high-risk patients.

Conclusion

In conclusion, our study indicates that gadofluorine-M–enhanced MRI is a reliable noninvasive tool to identify atherosclerotic plaques with features of vulnerability in vivo. We found good correlation between atherosclerotic plaque enhancement and neovessel density in an animal model of atherosclerosis. Our study provided direct evidence of gadofluorine-M colocalization with neovessel-rich regions, suggesting that one of the mechanisms for enhancement is mediated by plaque neovascularization. These findings may further encourage clinical development of gadofluorine-M–enhanced MRI for in vivo detection of vulnerable or high-risk plaques and potentially lead to improvement in noninvasive risk stratification of patients.